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Sample records for vaginal epithelial cells

  1. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

    PubMed

    Ito, Takuji; Bai, Tao; Tanaka, Tetsuji; Yoshida, Kenji; Ueyama, Takashi; Miyajima, Masayasu; Negishi, Takayuki; Kawasaki, Takahiko; Takamatsu, Hyota; Kikutani, Hitoshi; Kumanogoh, Atsushi; Yukawa, Kazunori

    2015-02-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild‑type (WT) mice. Administration of β‑estradiol to infant Sema4D‑deficient (Sema4D‑/‑) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β‑estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin‑B1, was examined as well as the level of apoptosis in the vaginal epithelia of five‑week‑old WT and Sema4D‑/‑ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin‑B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase‑3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five‑week‑old Sema4D‑/‑ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D. The experimental reduction of plexin‑B1 expression in vaginal epithelial cells demonstrated the integral role of plexin‑B1 in Sema4D‑induced apoptotic cell death. These results suggest a non‑redundant role of Sema4D in the postnatal tissue remodeling process in five‑week‑old BALB/c mice, which involves the induction of vaginal epithelial cell apoptosis through Sema4D binding to plexin‑B1. PMID:25351707

  2. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling

    PubMed Central

    ITO, TAKUJI; BAI, TAO; TANAKA, TETSUJI; YOSHIDA, KENJI; UEYAMA, TAKASHI; MIYAJIMA, MASAYASU; NEGISHI, TAKAYUKI; KAWASAKI, TAKAHIKO; TAKAMATSU, HYOTA; KIKUTANI, HITOSHI; KUMANOGOH, ATSUSHI; YUKAWA, KAZUNORI

    2015-01-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild-type (WT) mice. Administration of ?-estradiol to infant Sema4D-deficient (Sema4D?/?) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same ?-estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin-B1, was examined as well as the level of apoptosis in the vaginal epithelia of five-week-old WT and Sema4D?/? mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin-B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase-3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five-week-old Sema4D?/? mice compared with WT mice. The addition of recombinant Sema4D to Sema4D?/? vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis-inducing activity of Sema4D. The experimental reduction of plexin-B1 expression in vaginal epithelial cells demonstrated the integral role of plexin-B1 in Sema4D-induced apoptotic cell death. These results suggest a non-redundant role of Sema4D in the postnatal tissue remodeling process in five-week-old BALB/c mice, which involves the induction of vaginal epithelial cell apoptosis through Sema4D binding to plexin-B1. PMID:25351707

  3. Analysis of the Oxidative Stress Status in Nonspecific Vaginitis and Its Role in Vaginal Epithelial Cells Apoptosis

    PubMed Central

    Chen, Zhaojie; Zhang, Zhen; Zhang, Haiyan; Xie, Beibei

    2015-01-01

    Nonspecific vaginitis (NSV), also named bacterial vaginosis, is one of the most common genital system diseases in women during their reproductive years. The specific pathogenic mechanism of NSV is not clear yet. Upon the balance alteration, large amount of reactive oxidant species (ROS) is generated and accumulated in the genital tract, and thus resulting in oxidative stress, which has been reported to be an important trigger of mitochondrial pathway cell apoptosis. In this study, the antioxidant secretion level and antioxidant enzyme activity in the vaginal discharge were evaluated to analyze the oxidative status in the vaginal tract of NSV patients. The effect of oxidative stress on the vaginal mucosa epithelial cell apoptosis was then studied. The role of oxidative stress on NSV development was uncovered; thus open new direction for the prevention and treatment of NSV by providing antiradical agents was revealed. PMID:26558281

  4. Growth of Normal Mouse Vaginal Epithelial Cells in and on Collagen Gels

    NASA Astrophysics Data System (ADS)

    Iguchi, Taisen; Uchima, Francis-Dean A.; Ostrander, Patricia L.; Bern, Howard A.

    1983-06-01

    Sustained growth in primary culture of vaginal epithelial cells from ovariectomized adult BALB/cCrg1 mice embedded within or seeded on collagen gel matrix was achieved in a serum-free medium composed of Ham's F-12 medium/Dulbecco's modified Eagle's medium, 1:1 (vol/vol), supplemented with insulin, bovine serum albumin fraction V, epidermal growth factor, cholera toxin, and transferrin. Three-dimensional growth of vaginal epithelial cells occurred inside the collagen gel matrix. Cell numbers increased 4- to 8-fold in collagen gel and about 4-fold on collagen gel after 9-10 days in culture. The effect of 17β -estradiol (0.00018-180 nM in gel or 0.018-180 nM on gel) and diethylstilbestrol (DES; 0.0186-186 nM in gel) on the growth of vaginal epithelial cells was examined. The addition of estrogen did not enhance the growth of vaginal epithelial cells during this time period either in the complete medium or in a suboptimal medium. Cultures on floating collagen gels in the serum-free medium are composed of 1-3 cell layers with superficial cornification. Estrogen does not appear to be a direct mitogen for vaginal epithelial cells, at least in this system.

  5. Vaginal epithelial cells regulate membrane adhesiveness to co-ordinate bacterial adhesion.

    PubMed

    Younes, Jessica A; Klappe, Karin; Kok, Jan Willem; Busscher, Henk J; Reid, Gregor; van der Mei, Henny C

    2016-04-01

    Vaginal epithelium is colonized by different bacterial strains and species. The bacterial composition of vaginal biofilms controls the balance between health and disease. Little is known about the relative contribution of the epithelial and bacterial cell surfaces to bacterial adhesion and whether and how adhesion is regulated over cell membrane regions. Here, we show that bacterial adhesion forces with cell membrane regions not located above the nucleus are stronger than with regions above the nucleus both for vaginal pathogens and different commensal and probiotic lactobacillus strains involved in health. Importantly, adhesion force ratios over membrane regions away from and above the nucleus coincided with the ratios between numbers of adhering bacteria over both regions. Bacterial adhesion forces were dramatically decreased by depleting the epithelial cell membrane of cholesterol or sub-membrane cortical actin. Thus, epithelial cells can regulate membrane regions to which bacterial adhesion is discouraged, possibly to protect the nucleus. PMID:26477544

  6. Commensal Bacteria Modulate Innate Immune Responses of Vaginal Epithelial Cell Multilayer Cultures

    PubMed Central

    Rose, William A.; McGowin, Chris L.; Spagnuolo, Rae Ann; Eaves-Pyles, Tonyia D.; Popov, Vsevolod L.; Pyles, Richard B.

    2012-01-01

    The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives. PMID:22412914

  7. The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro

    PubMed Central

    Manczinger, Mt; Bocsik, Alexandra; Kocsis, Gabriella F.; Vrs, Andrea; Heged?s, Zoltn; rdgh, Lilla; Kondorosi, va; Marton, Annamria; Vzler, Csaba; Tubak, Vilmos; Deli, Mria; Kemny, Lajos; Nagy, Istvn; Lakatos, Lrnt

    2015-01-01

    To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis. PMID:26366412

  8. The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro.

    PubMed

    Manczinger, Mt; Bocsik, Alexandra; Kocsis, Gabriella F; Vrs, Andrea; Heged?s, Zoltn; rdgh, Lilla; Kondorosi, va; Marton, Annamria; Vzler, Csaba; Tubak, Vilmos; Deli, Mria; Kemny, Lajos; Nagy, Istvn; Lakatos, Lrnt

    2015-01-01

    To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis. PMID:26366412

  9. Innate Immunity in the Vagina (Part I): Estradiol Inhibits HBD2 and Elafin Secretion by Human Vaginal Epithelial Cells

    PubMed Central

    Patel, Mickey V.; Fahey, John V.; Rossoll, Richard M.; Wira, Charles R.

    2013-01-01

    Problem Vaginal epithelial cells (VEC) are the first line of defense against incoming pathogens in the female reproductive tract. Their ability to produce the anti-HIV molecules elafin and HBD2 under hormonal stimulation is unknown. Method of study Vaginal epithelial cells were recovered using a menstrual cup and cultured overnight prior to treatment with estradiol (E2), progesterone (P4) or a panel of selective estrogen response modulators (SERMs). Conditioned media were recovered and analyzed for protein concentration and anti-HIV activity. Results E2 significantly decreased the secretion of HBD2 and elafin by VEC over 48 hrs, while P4 and the SERMs (tamoxifen, PHTTP, ICI or Y134) had no effect. VEC conditioned media from E2-treated cells had no anti-HIV activity, while that from E2/P4-treated cells significantly inhibited HIV-BaL infection. Conclusion The menstrual cup allows for effective recovery of primary VEC. Their production of HBD2 and elafin is sensitive to E2, suggesting that innate immune protection varies in the vagina across the menstrual cycle. PMID:23398087

  10. Protocol for Examining Human Vaginal Epithelial Cell Signaling in Response to Staphylococcal Superantigens.

    PubMed

    Breshears, Laura M; Peterson, Marnie L

    2016-01-01

    A detailed investigation of eukaryotic signaling pathways affected by bacterial products is key to our understanding of host-pathogen interactions. Cytokine expression appears to be an important initial host cell response to many bacterial products, including the Staphylococcus aureus superantigens (SAgs). While much is understood about how SAgs signal to immune cells, very little is known about the specific cellular pathways activated by SAgs on nonimmune cells such as those of the epithelium. Here, we describe methods for analyzing SAg signaling in cultured epithelial cells, which may be extrapolated to the analysis of signaling pathways induced by other bacterial ligands on a variety of cell types. PMID:26676045

  11. Bone marrow mesenchymal stem cells could acquire the phenotypes of epithelial cells and accelerate vaginal reconstruction combined with small intestinal submucosa.

    PubMed

    Li, Yanan; Liu, Fangfang; Zhang, Zhiqiang; Zhang, Mingle; Cao, Shanjin; Li, Yachai; Zhang, Lin; Huang, Xianghua; Xu, Yanfang

    2015-11-01

    Grafting material for vaginal reconstruction commonly includes the bowel, peritoneum, skin, and amniotic membrane. Bone marrow mesenchymal stem cells (MSCs) have the potential of multilineage differentiation into a variety of cells and have been widely explored in tissue engineering. In the current study, we examined whether MSCs could be differentiated to vaginal epithelial cells (VECs) upon co-culturing with VECs. We also examined whether Wnt/β-catenin signaling pathway is implicated in such differentiation. Co-culture of MSCs with VECs using a transwell insert system (with no direct contact) induced the expression of VECs marker AE1/AE3 in MSCs. MSCs combined with small intestinal submucosa (SIS) scaffold were implanted in place of the native vagina in rats to observe the implications for vaginal reconstruction in vivo. Anatomic repair of neovagina was assessed by histological staining for H/E and Masson's Trichrome. GSK-3β and β-catenin, main members of Wnt/β-catenin signaling pathway, in MSCs were increased upon co-culturing with VECs. Exposure of co-cultured MSCs to a Wnt/β-catenin signaling activator, lithium chloride (LiCl, 20 µM) increased phosphorylated GSK-3β and β-catenin and enhanced expression of AE1/AE3. In vivo-grafted cells displayed significant matrix infiltration and expressed epithelial markers in neovagina. These findings suggest that MSCs could acquire the phenotype of VECs when co-cultured with VECs, possibly via activation of Wnt/β-catenin signaling. MSCs provide an alternative cell source for potential use in vaginal tissue engineering. PMID:26018040

  12. Vaginitis.

    PubMed

    Hainer, Barry L; Gibson, Maria V

    2011-04-01

    Bacterial vaginosis, trichomoniasis, and vulvovaginal candidiasis are the most common infectious causes of vaginitis. Bacterial vaginosis occurs when the normal lactobacilli of the vagina are replaced by mostly anaerobic bacteria. Diagnosis is commonly made using the Amsel criteria, which include vaginal pH greater than 4.5, positive whiff test, milky discharge, and the presence of clue cells on microscopic examination of vaginal fluid. Oral and topical clindamycin and metronidazole are equally effective at eradicating bacterial vaginosis. Symptoms and signs of trichomoniasis are not specific; diagnosis by microscopy is more reliable. Features of trichomoniasis are trichomonads seen microscopically in saline, more leukocytes than epithelial cells, positive whiff test, and vaginal pH greater than 5.4. Any nitroimidazole drug (e.g., metronidazole) given orally as a single dose or over a longer period resolves 90 percent of trichomoniasis cases. Sex partners should be treated simultaneously. Most patients with vulvovaginal candidiasis are diagnosed by the presence of vulvar inflammation plus vaginal discharge or with microscopic examination of vaginal secretions in 10 percent potassium hydroxide solution. Vaginal pH is usually normal (4.0 to 4.5). Vulvovaginal candidiasis should be treated with one of many topical or oral antifungals, which appear to be equally effective. Rapid point-of-care tests are available to aid in accurate diagnosis of infectious vaginitis. Atrophic vaginitis, a form of vaginitis caused by estrogen deficiency, produces symptoms of vaginal dryness, itching, irritation, discharge, and dyspareunia. Both systemic and topical estrogen treatments are effective. Allergic and irritant contact forms of vaginitis can also occur. PMID:21524046

  13. Mannose-binding lectin is produced by vaginal epithelial cells and its level in the vaginal fluid is influenced by progesterone.

    PubMed

    Bulla, R; De Seta, F; Radillo, O; Agostinis, C; Durigutto, P; Pellis, V; De Santo, D; Crovella, S; Tedesco, F

    2010-01-01

    Mannose-binding lectin (MBL) is a recognition molecule of the complement (C) system and binds to carbohydrate ligands present on a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL has been detected in the cervico-vaginal cavity where it can provide a first-line defence against infectious agents colonizing the lower tract of the reproductive system. Analysis of the cervico-vaginal lavage (CVL) obtained from 11 normal cycling women at different phases of the menstrual cycle revealed increased levels of MBL in the secretive phase. Part of this MBL derives from the circulation as indicated by the presence of transferrin in CVL tested as a marker of vascular and tissue permeability. The local synthesis of MBL is suggested by the finding that its level is substantially higher than that of transferrin in the secretive phase. The contribution of endometrium is negligible since the MBL level did not change before and after hysterectomy. RT-PCR and in situ RT-PCR analysis showed that the vaginal tissue, and in particular the basal layer of the epithelium, is a source of MBL which binds to the basal membrane and to cells of the outer layers of the epithelium. In conclusion, we have shown that MBL detected in CVL derives both from plasma as result of transudation and from local synthesis and its level is progesterone dependent increasing in the secretive phase of the menstrual cycle. PMID:20728220

  14. A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

    PubMed Central

    Mundodi, V; Kucknoor, AS; Chang, T-H; Alderete, JF

    2006-01-01

    Background Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis. Results An IgA mAb called 6B8 was isolated from a library of mAbs reactive to surface proteins of T. vaginalis. The 6B8 mAb recognized a 44-kDa protein (TV44) by immunoblot analysis, and a full-length cDNA clone encoded a protein of 438 amino acids. Southern analysis revealed the gene (tv44) of T. vaginalis to be single copy. The tv44 gene was down-regulated at both the transcriptional and translational levels in iron-depleted trichomonads as well as in parasites after contact with immortalized MS-74 vaginal epithelial cells (VECs). Immunofluorescence on non-permeabilized organisms confirmed surface localization of TV44, and the intensity of fluorescence was reduced after parasite adherence to VECs. Lastly, an identical protein and gene were present in Tritrichomonas foetus and Trichomonas tenax. Conclusion This is the first report of a T. vaginalis gene (tv44) encoding a surface protein (TV44) reactive with an IgA mAb, and both gene and protein were conserved in human and bovine trichomonads. Further, TV44 is independently down-regulated in expression and surface placement by iron and contact with VECs. TV44 is another member of T. vaginalis genes that are regulated by at least two independent signaling mechanisms involving iron and contact with VECs. PMID:16448556

  15. Reference Gene Selection for qPCR Is Dependent on Cell Type Rather than Treatment in Colonic and Vaginal Human Epithelial Cell Lines

    PubMed Central

    Jacobsen, Annette V.; Yemaneab, Bisrat T.; Jass, Jana; Scherbak, Nikolai

    2014-01-01

    The ability of commensal bacteria to influence gene expression in host cells under the influence of pathogenic bacteria has previously been demonstrated, however the extent of this interaction is important for understanding how bacteria can be used as probiotics. Real-time quantitative polymerase chain reaction is the most sensitive tool for evaluating relative changes to gene expression levels. However as a result of its sensitivity an appropriate method of normalisation should be used to account for any variation incurred in preparatory experimental procedures. These variations may result from differences in the amount of starting material, quality of extracted RNA, or in the efficiency of the reverse transcriptase or polymerase enzymes. Selection of an endogenous control gene is the preferred method of normalisation, and ideally a proper validation of the gene's appropriateness for the study in question should be performed. In this study we used quantitative polymerase chain reaction data and applied four different algorithms (geNorm, BestKeeper, NormFinder, and comparative ?Cq) to evaluate eleven different genes as to their suitability as endogenous controls for use in studies involving colonic (HT-29) and vaginal (VK2/E6E7) human mucosal epithelial cells treated with probiotic and pathogenic bacteria. We found phosphoglycerate kinase 1 to be most appropriate for HT-29 cells, and ribosomal protein large P0 to be the best choice for VK2/E6E7 cells. We also showed that use of less stable reference genes can lead to less accurate quantification of expression levels of gene of interest (GOI) and also can result in decreased statistical significance for GOI expression levels when compared to control. Additionally, we found the cell type being analysed had greater influence on reference gene selection than the treatment performed. This study provides recommendations for stable endogenous control genes for use in further studies involving colonic and vaginal cell lines after bacterial challenge. PMID:25526394

  16. Vaginal Epithelial Cell-Derived S100 Alarmins Induced by Candida albicans via Pattern Recognition Receptor Interactions Are Sufficient but Not Necessary for the Acute Neutrophil Response during Experimental Vaginal Candidiasis

    PubMed Central

    Yano, Junko; Palmer, Glen E.; Eberle, Karen E.; Peters, Brian M.; Vogl, Thomas; McKenzie, Andrew N.

    2014-01-01

    Vulvovaginal candidiasis (VVC), caused by Candida albicans, affects women worldwide. Animal and clinical studies suggest that the immunopathogenic inflammatory condition of VVC is initiated by S100 alarmins in response to C. albicans, which stimulate polymorphonuclear neutrophil (PMN) migration to the vagina. The purpose of this study was to extend previous in vitro data and determine the requirement for the alarmin S100A8 in the PMN response and to evaluate pattern recognition receptors (PRRs) that initiate the response. For the former, PMN migration was evaluated in vitro or in vivo in the presence or absence of S100 alarmins initiated by several approaches. For the latter, vaginal epithelial cells were evaluated for PRR expression and C. albicans-induced S100A8 and S100A9 mRNAs, followed by evaluation of the PMN response in inoculated PRR-deficient mice. Results revealed that, consistent with previously reported in vitro data, eukaryote-derived S100A8, but not prokaryote-derived recombinant S100A8, induced significant PMN chemotaxis in vivo. Conversely, a lack of biologically active S100A8 alarmin, achieved by antibody neutralization or by using S100A9?/? mice, had no effect on the PMN response in vivo. In PRR analyses, whereas Toll-like receptor 4 (TLR4)- and SIGNR1-deficient vaginal epithelial cells showed a dramatic reduction in C. albicans-induced S100A8/S100A9 mRNAs in vitro, inoculated mice deficient in these PRRs showed PMN migration similar to that in wild-type controls. These results suggest that S100A8 alarmin is sufficient, but not necessary, to induce PMN migration during VVC and that the vaginal PMN response to C. albicans involves PRRs in addition to SIGNR1 and TLR4, or other induction pathways. PMID:24478092

  17. Comparative in vitro sensitivities of human immune cell lines, vaginal and cervical epithelial cell lines, and primary cells to candidate microbicides nonoxynol 9, C31G, and sodium dodecyl sulfate.

    PubMed

    Krebs, Fred C; Miller, Shendra R; Catalone, Bradley J; Fichorova, Raina; Anderson, Deborah; Malamud, Daniel; Howett, Mary K; Wigdahl, Brian

    2002-07-01

    In experiments to assess the in vitro impact of the candidate microbicides nonoxynol 9 (N-9), C31G, and sodium dodecyl sulfate (SDS) on human immune and epithelial cell viability, cell lines and primary cell populations of lymphocytic and monocytic origin were generally shown to be equally sensitive to exposures ranging from 10 min to 48 h. However, U-937 cells were more sensitive to N-9 and C31G after 48 h than were primary monocyte-derived macrophages. Cytokine activation of monocytes and lymphocytes had no effect on cell viability following exposure to these microbicidal compounds. Primary and passaged vaginal epithelial cultures and cell lines differed in sensitivity to N-9 and C31G but not SDS. These studies provide a foundation for in vitro experiments in which cell lines of human immune and epithelial origin can be used as suitable surrogates for primary cells to further investigate the effects of microbicides on cell metabolism, membrane composition, and integrity and the effects of cell type, proliferation, and differentiation on microbicide sensitivity. PMID:12069993

  18. Comparative In Vitro Sensitivities of Human Immune Cell Lines, Vaginal and Cervical Epithelial Cell Lines, and Primary Cells to Candidate Microbicides Nonoxynol 9, C31G, and Sodium Dodecyl Sulfate

    PubMed Central

    Krebs, Fred C.; Miller, Shendra R.; Catalone, Bradley J.; Fichorova, Raina; Anderson, Deborah; Malamud, Daniel; Howett, Mary K.; Wigdahl, Brian

    2002-01-01

    In experiments to assess the in vitro impact of the candidate microbicides nonoxynol 9 (N-9), C31G, and sodium dodecyl sulfate (SDS) on human immune and epithelial cell viability, cell lines and primary cell populations of lymphocytic and monocytic origin were generally shown to be equally sensitive to exposures ranging from 10 min to 48 h. However, U-937 cells were more sensitive to N-9 and C31G after 48 h than were primary monocyte-derived macrophages. Cytokine activation of monocytes and lymphocytes had no effect on cell viability following exposure to these microbicidal compounds. Primary and passaged vaginal epithelial cultures and cell lines differed in sensitivity to N-9 and C31G but not SDS. These studies provide a foundation for in vitro experiments in which cell lines of human immune and epithelial origin can be used as suitable surrogates for primary cells to further investigate the effects of microbicides on cell metabolism, membrane composition, and integrity and the effects of cell type, proliferation, and differentiation on microbicide sensitivity. PMID:12069993

  19. Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

    PubMed Central

    Chatterjee, Aparajita; Ratner, Daniel M.; Ryan, Christopher M.; Johnson, Patricia J.; O’Keefe, Barry R.; Secor, W. Evan; Anderson, Deborah J.; Robbins, Phillips W.; Samuelson, John

    2015-01-01

    Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas. PMID:26252012

  20. Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model.

    PubMed

    Chatterjee, Aparajita; Ratner, Daniel M; Ryan, Christopher M; Johnson, Patricia J; O'Keefe, Barry R; Secor, W Evan; Anderson, Deborah J; Robbins, Phillips W; Samuelson, John

    2015-01-01

    Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas. PMID:26252012

  1. Cultivated Vaginal Microbiomes Alter HIV-1 Infection and Antiretroviral Efficacy in Colonized Epithelial Multilayer Cultures

    PubMed Central

    Pyles, Richard B.; Vincent, Kathleen L.; Baum, Marc M.; Elsom, Barry; Miller, Aaron L.; Maxwell, Carrie; Eaves-Pyles, Tonyia D.; Li, Guangyu; Popov, Vsevolod L.; Nusbaum, Rebecca J.; Ferguson, Monique R.

    2014-01-01

    There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral. PMID:24676219

  2. Desquamative inflammatory vaginitis.

    PubMed

    Reichman, Orna; Sobel, Jack

    2014-10-01

    Desquamative inflammatory vaginitis (DIV) is an uncommon form of chronic purulent vaginitis. It occurs mainly in Caucasians with a peak occurrence in the perimenopause. Symptoms and signs are nonspecific; DIV is a diagnosis of exclusion, and other causes of purulent vaginitis should be excluded. The main symptoms include purulent discharge, vestibulo-vaginal irritation, and dyspareunia. Examination of vaginal walls shows signs of inflammation with increased erythema and petechiae. Through microscopy (wet mount) of the vaginal secretions, DIV is defined by an increase in inflammatory cells and parabasal epithelial cells (immature squamous cells). Vaginal flora is abnormal and pH is always elevated above 4.5. Although etiology and pathogenesis remain unknown, the favorable response to anti-inflammatory agents suggests that the etiology is immune mediated. Either local vaginal clindamycin or vaginal corticosteroids are adequate treatment. As a chronic condition, maintenance treatment should be considered as relapse is common. PMID:25132275

  3. Vaginal Microbiome and Epithelial Gene Array in Post-Menopausal Women with Moderate to Severe Dryness

    PubMed Central

    Hammond, Jo-Anne; McMillan, Amy; Vongsa, Rebecca; Koenig, David; Gloor, Gregory B.; Reid, Gregor

    2011-01-01

    After menopause, many women experience vaginal dryness and atrophy of tissue, often attributed to the loss of estrogen. An understudied aspect of vaginal health in women who experience dryness due to atrophy is the role of the resident microbes. It is known that the microbiota has an important role in healthy vaginal homeostasis, including maintaining the pH balance and excluding pathogens. The objectives of this study were twofold: first to identify the microbiome of post-menopausal women with and without vaginal dryness and symptoms of atrophy; and secondly to examine any differences in epithelial gene expression associated with atrophy. The vaginal microbiome of 32 post-menopausal women was profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Sixteen subjects were selected for follow-up sampling every two weeks for 10 weeks. In addition, 10 epithelial RNA samples (6 healthy and 4 experiencing vaginal dryness) were acquired for gene expression analysis by Affymetrix Human Gene array. The microbiota abundance profiles were relatively stable over 10 weeks compared to previously published data on premenopausal women. There was an inverse correlation between Lactobacillus ratio and dryness and an increased bacterial diversity in women experiencing moderate to severe vaginal dryness. In healthy participants, Lactobacillus iners and L. crispatus were generally the most abundant, countering the long-held view that lactobacilli are absent or depleted in menopause. Vaginal dryness and atrophy were associated with down-regulation of human genes involved in maintenance of epithelial structure and barrier function, while those associated with inflammation were up-regulated consistent with the adverse clinical presentation. PMID:22073175

  4. Automated segmentation algorithm for detection of changes in vaginal epithelial morphology using optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Chitchian, Shahab; Vincent, Kathleen L.; Vargas, Gracie; Motamedi, Massoud

    2012-11-01

    We have explored the use of optical coherence tomography (OCT) as a noninvasive tool for assessing the toxicity of topical microbicides, products used to prevent HIV, by monitoring the integrity of the vaginal epithelium. A novel feature-based segmentation algorithm using a nearest-neighbor classifier was developed to monitor changes in the morphology of vaginal epithelium. The two-step automated algorithm yielded OCT images with a clearly defined epithelial layer, enabling differentiation of normal and damaged tissue. The algorithm was robust in that it was able to discriminate the epithelial layer from underlying stroma as well as residual microbicide product on the surface. This segmentation technique for OCT images has the potential to be readily adaptable to the clinical setting for noninvasively defining the boundaries of the epithelium, enabling quantifiable assessment of microbicide-induced damage in vaginal tissue.

  5. Spindle Cell Epithelioma: A Rare Vaginal Tumor A Clinico Pathologic Report

    PubMed Central

    K., Nivedita; Sowmya; Shanthini, Fatima

    2013-01-01

    Spindle cell epithelioma is a very rare benign tumour of the vagina, which contains epithelial and mesenchymal components and co-expresses the markers for both. It has its origin in the epithelial cells of the remnants of the vestibular gland. The presence of glandular structures and the pattern of immunostaining, help in the differentiation of these tumours from the other common vaginal tumours. PMID:24086899

  6. Vaginitis

    MedlinePLUS

    ... are the treatments? Are there complications? Does it affect pregnancy? How is it prevented? NICHD Research Information Clinical Trials Resources and Publications Vaginitis: Condition Information Skip sharing on social media links Share this: Page Content What is vaginitis? ...

  7. Epithelial Cell Adhesion Molecule

    PubMed Central

    Trzpis, Monika; McLaughlin, Pamela M.J.; de Leij, Lou M.F.H.; Harmsen, Martin C.

    2007-01-01

    The epithelial cell adhesion molecule (EpCAM, CD326) is a glycoprotein of ?40 kd that was originally identified as a marker for carcinoma, attributable to its high expression on rapidly proliferating tumors of epithelial origin. Normal epithelia express EpCAM at a variable but generally lower level than carcinoma cells. In early studies, EpCAM was proposed to be a cell-cell adhesion molecule. However, recent insights revealed a more versatile role for EpCAM that is not limited only to cell adhesion but includes diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EpCAM may actually prevent cell-cell adhesion. Here, we provide a comprehensive review of the current knowledge on EpCAM biology in relation to other cell adhesion molecules. We discuss the implications of the newly identified functions of EpCAM in view of its prognostic relevance in carcinoma, inflammatory pathophysiology, and tissue development and regeneration as well as its role in normal epithelial homeostasis. PMID:17600130

  8. Microbial adherence to vulvar epithelial cells.

    PubMed

    Bibel, D J; Aly, R; Lahti, L; Shinefield, H R; Maibach, H I

    1987-02-01

    Under uniform experimental conditions, different degrees of selective attachment of Staphylococcus aureus and Candida albicans to epithelial cells of the labium majus, the labium minus, and the vagina were compared and contrasted with those found in studies with cells of the buccal and nasal mucosa and forearm skin by a novel analysis of adherence density. For both micro-organisms, the larger, rougher cells of the labium majus gave the highest adherence scores matched only by the interaction of S. aureus with fully keratinised nasal epithelial cells. Increasing acidity to pH 3.5 enhanced microbial adherence to vaginal cells. Menstruation also influenced attachment; highest densities were reached between the third and fourth weeks of the cycle. Autogenous ribitol teichoic acid was found to block the attachment of S. aureus to labium majus and labium minus cells by 76% and 81% respectively, and to vaginal cells by 66%. Adherence is considered to be an important attribute of vulvar ecology and may be a determinant of infectious disease. PMID:3546698

  9. NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

    PubMed Central

    Luci, Carmelo; Bekri, Selma; Bihl, Franck; Pini, Jonathan; Bourdely, Pierre; Nouhen, Kelly; Malgogne, Angélique; Walzer, Thierry; Braud, Véronique M.; Anjuère, Fabienne

    2015-01-01

    Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses. PMID:26630176

  10. Intestinal Epithelial Cells In Vitro

    PubMed Central

    Dombkowski, Alan A.; Stemmer, Paul M.; Parker, Graham C.

    2010-01-01

    Recent advances in the biology of stem cells has resulted in significant interest in the development of normal epithelial cell lines from the intestinal mucosa, both to exploit the therapeutic potential of stem cells in tissue regeneration and to develop treatment models of degenerative disorders of the digestive tract. However, the difficulty of propagating cell lines of normal intestinal epithelium has impeded research into the molecular mechanisms underlying differentiation of stem/progenitor cells into the various intestinal lineages. Several short-term organ/organoid and epithelial cell culture models have been described. There is a dearth of long-term epithelial and/or stem cell cultures of intestine. With an expanding role of stem cells in the treatment of degenerative disorders, there is a critical need for additional efforts to develop in vitro models of stem/progenitor epithelial cells of intestine. The objective of this review is to recapitulate the current status of technologies and knowledge for in vitro propagation of intestinal epithelial cells, markers of the intestinal stem cells, and gene and protein expression profiles of the intestinal cellular differentiation. PMID:19580443

  11. 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

  12. A physical method for separating spermatozoa from epithelial cells in sexual assault evidence.

    PubMed

    Chen, J; Kobilinsky, L; Wolosin, D; Shaler, R; Baum, H

    1998-01-01

    The analysis of genetic markers for the purpose of individualization of semen specimens is extremely important in cases of sexual abuse and assault. The serological analysis of sexual assault evidence can sometimes be complicated because stains are often composed of a mixture of spermatozoa, vaginal epithelial cells and white and red blood cells. A filtration method has been developed to cleanly separate spermatozoa from epithelial cells based upon differences in size and shape. Nylon mesh filters of the appropriate pore size can be used to separate the smaller oval shaped spermatozoal cells from the larger and flatter epithelial cells. The former pass freely through the membrane while the latter are retained on the filter. In this study, cell separation was demonstrated by (a) microscopic observation of stained cells, (b) amplified fragment length polymorphism analysis of DNA obtained from separated cells. The results of these analyses indicate that: (1) Approximately 70% of spermatozoa in the mixed cell sample will penetrate the 10 microns pore size filter, (2) Only about 1-2% of intact epithelial cells will do so, and (3) A small number of nuclei from spontaneously lysed epithelial cells will cross the filter. Experimental results using mixtures of spermatozoa and vaginal epithelial cells prepared in different ratios support the conclusion that the filtration process is an efficient and reliable method to separate spermatozoa from epithelial cells in casework specimens for subsequent DNA analysis. PMID:9456531

  13. 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.

  14. Kidney epithelial cells.

    PubMed

    Smith, Peter L; Buffington, Deborah A; Humes, H David

    2006-01-01

    Kidney tubules are an essential component of an organism's blood clearance mechanism, recovering essential metabolites from glomerular filtration by active transport. Tubules are subject to injury, usually as the result of ischemia-reperfusion events that damage the polarized tubular cell layer that coats the tubule basement membrane, causing dysfunction and necrosis that is often associated with acute renal failure. However, tubules are capable of self-repair, forming new proximal tubular cells to replace failing or necrotic cells. The origin of the progenitor cells that give rise to new tubular cells is unknown. At one extreme, it is possible that all or a fraction of tubular cells can undergo a form of dedifferentiation and subsequent mitosis to form new tubular cells, or alternatively, it is possible that tubular regeneration follows the stem cell/transit-amplifying cell paradigm described for more rapidly regenerating organ systems. Regardless of the mechanism employed to generate new tubular cells, human tubular cells are readily grown in primary cultures and can recapitulate many of the metabolic, endocrine, and immunological properties attributable to endogenous renal proximal tubules when engrafted into bioartificial devices. PMID:17141057

  15. A Rabbit Vaginal Cell-Derived Antimicrobial Peptide, RVFHb?P, Blocks Lipopolysaccharide-Mediated Inflammation in Human Vaginal Cells In Vitro ?

    PubMed Central

    Patgaonkar, Mandar S.; Sathe, Ameya; Selvaakumar, C.; Reddy, K. V. R.

    2011-01-01

    Antimicrobial peptides (AMPs) constitute a phylogenetically ancient form of innate immunity that provides host defense at various mucosal surfaces, including the vagina. Recently, we have identified one such AMP, rabbit vaginal fluid hemoglobin alpha peptide (RVFHb?P), from the vaginal lavage of rabbits (Oryctolagus cuniculus). The recent demonstration of a protective role of this peptide in erythrocytes and vaginal cells led us to investigate (i) the lipopolysaccharide (LPS) interactive domain in RVFHb?P and (ii) whether RVFHb?P of rabbit origin modulates the cellular immune responses of another species (humans) in vitro. HeLa-S3, a human vaginal epithelial cell line (hVEC), was exposed to LPS alone (10 ?g/ml for 6 h), or LPS-induced cells were treated with RVFHb?P (70.45 ?M for 1 h) and cultured for 24 h, and the results obtained were compared with the medium control. We show here that RVFHb?P exerts an anti-inflammatory activity in hVECs, as suggested by the prevention of LPS-induced production of extracellular (supernatant) and intracellular (lysate) levels of cytokines (interleukin 6 [IL-6] and IL-1?) and chemokines (IL-8 and monocyte chemoattractant protein 1 [MCP-1]). The demonstration of Toll-like receptor 4 (TLR4) and NF-?B expression in hVECs and the observations of RVFHb?P suppression of human ?-defensin-1 (hBD1) mRNA expression further support the hypothesis of a genomic activity of RVFHb?P. Confocal microscopy and flow cytometry results demonstrate that RVFHb?P inhibits LPS-induced phagocytosis of Escherichia coli by macrophages. The chemotaxis studies performed using the Boyden chamber Transwell method showed the increased migration of U937 cells when supernatants of LPS-induced hVECs were used, and this effect was inhibited by RVFHb?P. In conclusion, our study proposes a novel explanation for the protective role of RVFHb?P in inflammation-associated infections, which not only may provide the new cellular targets for the screening of RVFHb?P ligands acting in the vaginal tissue but also has the potential to develop RVFHb?P as a therapeutic agent for reproductive tract infections. PMID:21865417

  16. Unique roles of estrogen-dependent Pten control in epithelial cell homeostasis of mouse vagina.

    PubMed

    Miyagawa, S; Sato, M; Sudo, T; Yamada, G; Iguchi, T

    2015-02-19

    Numerous studies support a role of phosphatase and tensin homolog deleted from chromosome 10 (Pten) as a tumor suppressor gene that controls epithelial cell homeostasis to prevent tumor formation. Mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen and provides a unique model for analyzing homeostasis of stratified squamous epithelia. We analyzed vaginal epithelium-specific Pten conditional knockout (CKO) mice to provide new insights into Pten/phosphoinositide-3-kinase (PI3K)/Akt function. The vaginal epithelium of ovariectomized (OVX) mice (control) was composed of 1-2 layers of cuboidal cells, whereas OVX CKO mice exhibited epithelial hyperplasia in the suprabasal cells with increased cell mass and mucin production. This is possibly due to misactivation of mammalian target of rapamycin and mitogen-activated protein kinase. Intriguingly, estrogen administration to OVX Pten CKO mice induced stratification and keratinized differentiation in the vaginal epithelium, as in estrogen-treated controls. We found that Pten is exclusively expressed in the suprabasal cells in the absence of estrogens, whereas estrogen administration induced Pten expression in the basal cells. This suggests that Pten acts to prevent excessive cell proliferation as in the case of other squamous tissues. Thus, Pten exhibits a dual role on the control of vaginal homeostasis, depending on whether estrogens are present or absent. Our results provide new insights into how Pten functions in tissue homeostasis. PMID:24632614

  17. 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

  18. Airway Epithelial Cell Migration Dynamics

    PubMed Central

    Legrand, Claire; Gilles, Christine; Zahm, Jean-Marie; Polette, Myriam; Buisson, Anne-Ccile; Kaplan, Herv; Birembaut, Philippe; Tournier, Jean-Marie

    1999-01-01

    Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, MMP-9 accumulated in migrating HBEC located at the leading edge of a wound and MMP-9 expression paralleled cell migration speed. MMP-9 accumulated through an actin-dependent pathway in the advancing lamellipodia of migrating cells and was subsequently found active in the extracellular matrix (ECM). Lamellipodia became anchored through primordial contacts established with type IV collagen. MMP-9 became amassed behind collagen IV where there were fewer cellECM contacts. Both collagen IV and MMP-9 were involved in cell migration because when cellcollagen IV interaction was blocked, cells spread slightly but did not migrate; and when MMP-9 activation was prevented, cells remained fixed on primordial contacts and did not advance at all. These observations suggest that MMP-9 controls the migration of repairing HBEC by remodeling the provisional ECM implicated in primordial contacts. PMID:10427102

  19. Epithelial cell contributions to intestinal immunity.

    PubMed

    Hooper, Lora V

    2015-01-01

    The epithelial surfaces of the mammalian intestine interface directly with the external environment and thus continuously encounter pathogenic bacteria, fungi, viruses, and parasites. The intestinal epithelium is also closely associated with complex communities of symbiotic microorganisms. Intestinal epithelial cells are thus faced with the unique challenge of directly interacting with enormous numbers of microbes that include both pathogens and symbionts. As a result, gut epithelia have evolved an array of strategies that contribute to host immunity. This chapter considers the various mechanisms used by epithelial cells to limit microbial invasion of host tissues, shape the composition of indigenous microbial communities, and coordinate the adaptive immune response to microorganisms. Study of intestinal epithelial cells has contributed fundamental insights into intestinal immune homeostasis and has revealed how impaired epithelial cell function can contribute to inflammatory disease. PMID:25727289

  20. Epithelial TRPV1 Signaling Accelerates Gingival Epithelial Cell Proliferation

    PubMed Central

    Takahashi, N.; Matsuda, Y.; Yamada, H.; Tabeta, K.; Nakajima, T.; Murakami, S.; Yamazaki, K.

    2014-01-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca2+ levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation. PMID:25266715

  1. Human glomerular epithelial cell proteoglycans

    SciTech Connect

    Thomas, G.J.; Jenner, L.; Mason, R.M.; Davies, M. )

    1990-04-01

    Proteoglycans synthesized by cultures of human glomerular epithelial cells have been isolated and characterized. Three types of heparan sulfate were detected. Heparan sulfate proteoglycan I (HSPG-I; Kav 6B 0.04) was found in the cell layer and medium and accounted for 12% of the total proteoglycans synthesized. HSPG-II (Kav 6B 0.25) accounted for 18% of the proteoglycans and was located in the medium and cell layer. A third population (9% of the proteoglycan population), heparan sulfate glycosaminoglycan (HS-GAG; Kav 6B 0.4-0.8), had properties consistent with single glycosaminoglycan chains or their fragments and was found only in the cell layer. HSPG-I and HSPG-II from the cell layer had hydrophobic properties; they were released from the cell layer by mild trypsin treatment. HS-GAG lacked these properties, consisted of low-molecular-mass heparan sulfate oligosaccharides, and were intracellular. HSPG-I and -II released to the medium lacked hydrophobic properties. The cells also produced three distinct types of chondroitin sulfates. The major species, chondroitin sulfate proteoglycan I (CSPG-I) eluted in the excluded volume of a Sepharose CL-6B column, accounted for 30% of the proteoglycans detected, and was found in both the cell layer and medium. Cell layer CSPG-I bound to octyl-Sepharose. It was released from the cell layer by mild trypsin treatment. CSPG-II (Kav 6B 0.1-0.23) accounted for 10% of the total 35S-labeled macromolecules and was found predominantly in the culture medium. A small amount of CS-GAG (Kav 6B 0.25-0.6) is present in the cell extract and like HS-GAG is intracellular. Pulse-chase experiments indicated that HSPG-I and -II and CSPG-I and -II are lost from the cell layer either by direct release into the medium or by internalization where they are metabolized to single glycosaminoglycan chains and subsequently to inorganic sulfate.

  2. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  3. Airway epithelial cell responses to ozone injury.

    PubMed Central

    Leikauf, G D; Simpson, L G; Santrock, J; Zhao, Q; Abbinante-Nissen, J; Zhou, S; Driscoll, K E

    1995-01-01

    The airway epithelial cells is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products--hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines--macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretory leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. PMID:7614953

  4. Evidence for a unique expression of CD4 on murine vaginal CD4+ cells

    PubMed Central

    Wormley, F L; Scott, M; Luo, W; Baker, M; Chaiban, J; Fidel, P L

    2000-01-01

    Mucosal cell-mediated immunity (CMI) by CD4+ T cells is postulated to be important for host defence against several vaginal pathogens. In addition to the recognized phenotypic distinctions of resident vaginal T lymphocytes, we recently provided evidence by fluorescence-activated cell sorter (FACS) that murine vaginal CD4+ T lymphocytes, are differentially recognized by two epitope-distinct anti-CD4 antibodies, suggesting that the CD4 protein on vaginal CD4+ cells is atypically expressed. In the present study, we confirm this by FACS and immunohistochemistry under non-denaturing conditions using two additional anti-CD4 antibodies. However, positive immunohistochemical staining of vaginal CD4+ cells under denaturing conditions revealed that the CD4 epitope in question is indeed present within the CD4 protein. Using reverse transcription polymerase chain reaction, amplification of CD3, T-cell receptor-β (TCR-β), and TCR-δ mRNA from lymph node and vaginal tissue, and CD4 mRNA from lymph node tissue was demonstrable. In contrast, amplification of CD4 mRNA from vaginal tissue, vaginal enriched lymphoid cells, or a purified (FACS-sorted) population of vaginal-specific CD4+ cells using two distinct primer sets was not demonstrable. Altogether, our results provide evidence that the CD4 protein on vaginal CD4+ T cells is conformationally distinct compared with its systemic counterpart, either as a result of a unique CD4 mRNA sequence or from a stable interaction of soluble CD4 with the surface of vaginal T cells. PMID:10929051

  5. [Streptococcus group B--association with Aerobic vaginitis and ability to human cell lines activation].

    PubMed

    Romanik, Ma?gorzata; Kafel, Joanna; Lagergrd, Teresa; Martirosian, Gayane

    2007-01-01

    The aim of this study was to estimate: the frequency of aerobic vaginitis, susceptibility of the GBS isolated from vagina of non-pregnant women with and without cervicitis to selected antibiotics and chemotherapeutics and the proinflammatory cytokines production by HeLa, THP-I, U - 937 cells after stimulation by vaginal GBS. Our results indicated low frequency of the aerobic vaginitis -4.5% among non-pregnant young women and ability of the vaginal GBS to release proinflammatory cytokines by human cell lines in vitro. PMID:17929406

  6. Vaginal laparoscopically assisted radical trachelectomy in cervical clear cell adenocarcinoma.

    PubMed

    Iacoponi, Sara; Diestro, Maria Dolores; Zapardiel, Ignacio; Serrano, Mara; Santiago, Javier De

    2013-01-01

    Adenocarcinoma of the cervix is a rare condition that has shown an increase in incidence, especially in the 20- to 34-year-old group. Adenocarcinoma represents about 5-10% of all tumours in this area, and, among these, the clear cell type accounts for 4-9%. This type of tumour affects mainly postmenopausal women but also occurs in young women with a history of prenatal exposure to diethylstilbestrol (DES). The prognosis for adenocarcinoma of the cervix is poor overall and worse for the clear cell variety. This article discusses a case of clear cell adenocarcinoma of the cervix, unrelated to intrauterine exposure to DES, in a woman of childbearing age who wished to preserve her fertility and was therefore treated by radical vaginal trachelectomy and pelvic lymphadenectomy. PMID:24244219

  7. Polarized sorting and trafficking in epithelial cells

    PubMed Central

    Cao, Xinwang; Surma, Michal A; Simons, Kai

    2012-01-01

    The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting. PMID:22525333

  8. The ontogeny of epithelial skin stem cells

    PubMed Central

    Tumbar, Tudorita

    2014-01-01

    Epithelial skin stem cells constitute an important model system for understanding the dynamics of stem cell emergence and behavior in an intact tissue. Recent published work defined discrete population of epithelial stem cells in the adult skin epithelium located in the lower hair follicle, isthmus, sebaceous gland and epidermis. Adult epidermal and hair follicle stem cells behave by a population deterministic model and adopt either one of two possible fates: (1) differentiate and be lost from the tissue or (2) expand symmetrically to self-renew. While the mechanisms controlling adult stem cell homeostasis begin to unravel, the embryonic development of epithelial skin stem cells is poorly understood. Few studies reported Sox9, Lgr6, and Runx1 expressed in subpopulations of cells in the embryonic hair placode that act as precursors of adult epithelial stem cells. Runx1 regulates a Wnt-mediated cross-talk between the nascent adult-type of hair follicle stem cells and their environment, which is essential for the stem cell timely emergence, proper maturation, long-term differentiation potential, and maintenance. The new data begin to define the basic dynamics and regulatory pathways governing the ontogeny of adult epithelial stem cells. PMID:22290419

  9. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    PubMed

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-01

    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. PMID:26859350

  10. Enhancement of Escherichia coli adherence to epithelial cells derived from estrogen-stimulated rats.

    PubMed Central

    Sobel, J D; Kaye, D

    1986-01-01

    The effect of exogenous estrogen administered to male and oophorectomized female rats was investigated with regard to in vitro adherence of eight uropathogenic strains of Escherichia coli to exfoliated bladder and vaginal epithelial cells. Uroepithelial cells obtained from estrogenized male and estrogenized oophorectomized female rats and vaginal cells obtained from estrogenized oophorectomized female rats demonstrated significantly enhanced (P less than 0.005) host cell avidity for E. coli attachment, irrespective of bacterial adhesin expressed, when compared with such cells from nonestrogenized male and female oophorectomized rats. These animal studies suggest that female reproductive hormones may contribute to urinary-tract infection in premenopausal females by enhancing susceptibility to E. coli colonization of uroepithelial cells. PMID:3522431

  11. 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. PMID:26367485

  12. Vaginal gel adsorption and retention by human vaginal cells: visual analysis by means of inorganic and organic markers.

    PubMed

    Braga, Pier Carlo; Dal Sasso, Monica; Spallino, Alessandra; Sturla, Carla; Culici, Maria

    2009-05-21

    To improve efficiency and prolong protection, modern gynecological preparations frequently incorporate polymeric molecules that add a certain degree of viscosity in order to increase adhesion with vaginal cells and prolong local delivery of active molecules. The aim of this study was to investigate the possibility of visualising the ability of a commercial medicated gynecological gel to bind to and be retained by human vaginal cells. The gel formulation included the essential oils of Thymus vulgaris and Eugenia cariophylla, which contain active molecules such as thymol and eugenol that are known to have useful antibacterial and antimycotic activities. The adherence of different dilutions of the gel to human vaginal cells was visualised by means of Nomarski interference contrast microscopy and scanning electron microscopy using ferric oxide particles and Escherichia coli as inorganic and organic markers, both of which made it possible to visualise the binding of the thin transparent layer of gel and the retaining effect, which was proportional to the degree of dilution. PMID:19429283

  13. Vaginal Cancer

    MedlinePLUS

    Vaginal cancer is a rare type of cancer. It is more common in women 60 and older. You are also more likely to get it if you have had a human ... test can find abnormal cells that may be cancer. Vaginal cancer can often be cured in its ...

  14. Biodegradable Film for the Targeted Delivery of siRNA-Loaded Nanoparticles to Vaginal Immune Cells.

    PubMed

    Gu, Jijin; Yang, Sidi; Ho, Emmanuel A

    2015-08-01

    The goal of this study was to develop and characterize a novel intravaginal film platform for targeted delivery of small interfering RNA (siRNA)-loaded nanoparticles (NP) to dendritic cells as a potential gene therapy for the prevention of sexually transmitted human immunodeficiency virus (HIV) infection. Poly(ethylene glycol) (PEG)-functionalized poly(D, L-lactic-co-glycolic acid) (PLGA)/polyethylenimine (PEI)/siRNA NP (siRNA-NP) were fabricated using a modified emulsion-solvent evaporation method and characterized for particle size, zeta potential, encapsulation efficiency (EE), and siRNA release. siRNA-NP were decorated with anti-HLA-DR antibody (siRNA-NP-Ab) for targeting delivery to HLA-DR+ dendritic cells (DCs) and homogeneously dispersed in a biodegradable film consisting of poly vinyl alcohol (PVA) and ?-carrageenan. The siRNA-NP-Ab-loaded film (siRNA-NP-Ab-film) was transparent, displayed suitable physicomechanical properties, and was noncytotoxic. Targeting activity was evaluated in a mucosal coculture model consisting of a vaginal epithelial monolayer (VK2/E6E7 cells) and differentiated KG-1 cells (HLA-DR+ DCs). siRNA-NP-Ab were rapidly released from the film and were able to penetrate the epithelial layer to be taken up by differentiated KG-1 cells. siRNA-NP-Ab demonstrated higher targeting activity and significantly higher knockdown of synaptosome-associated 23-kDa protein (SNAP-23) mRNA and protein when compared to siRNA-NP without antibody conjugation. Overall, these data suggest that our novel siRNA-NP-Ab-film may be a promising platform for preventing HIV infection within the female genital tract. PMID:26099315

  15. Epithelial-mesenchymal Transition and Cell Invasion

    PubMed Central

    Son, Hwajin

    2010-01-01

    Epithelial-mesenchymal transition (EMT) is a complex process in which epithelial cells acquire the characteristics of invasive mesenchymal cells. EMT has been implicated in cancer progression and metastasis as well as the formation of many tissues and organs during development. Epithelial cells undergoing EMT lose cell-cell adhesion structures and polarity, and rearrange their cytoskeletons. Several oncogenic pathways such as transforming growth factor (TGF) -?, Wnt, and Notch signaling pathways, have been shown to induce EMT. These pathways have activated transcription factors including Snail, Slug, and the ZEB family which work as transcriptional repressors of E-cadherin, thereby making epithelial cells motile and resistant to apoptosis. Mounting evidence shows that EMT is associated with cell invasion and tumor progression.In this review, we summarize the characteristic features of EMT, pathways leading to EMT, and the role of EMT in cell invasion. Three topics are addressed in this review: (1) Definition of EMT, (2) Signaling pathways leading to EMT, (3) Role of EMT in cell invasion. Understanding the role of EMT in cell invasion will provide valuable information for establishing strategies to develop anti-metastatic therapeutics which modulate malignant cellular processes mediated by EMT. PMID:24278531

  16. Determining Proportion of Exfoliative Vaginal Cell during Various Stages of Estrus Cycle Using Vaginal Cytology Techniques in Aceh Cattle

    PubMed Central

    Siregar, Tongku N.; Melia, Juli; Rohaya; Thasmi, Cut Nila; Masyitha, Dian; Wahyuni, Sri; Rosa, Juliana; Nurhafni; Panjaitan, Budianto; Herrialfian

    2016-01-01

    The aim of this study was to investigate the period of estrus cycle in aceh cattle, Indonesia, based on vaginal cytology techniques. Four healthy females of aceh cattle with average weight of 250–300 kg, age of 5–7 years, and body condition score of 3-4 were used. All cattle were subjected to ultrasonography analysis for the occurrence of corpus luteum before being synchronized using intramuscular injections of PGF2 alpha 25 mg. A vaginal swab was collected from aceh cattle, stained with Giemsa 10%, and observed microscopically. Period of estrus cycle was predicted from day 1 to day 24 after estrus synchronization was confirmed using ultrasonography analysis at the same day. The result showed that parabasal, intermediary, and superficial epithelium were found in the vaginal swabs collected from proestrus, metestrus, and diestrus aceh cattle. Proportions of these cells in the particular period of estrus cycle were 36.22, 32.62, and 31.16 (proestrus); 21.33, 32.58, and 46.09 (estrus); 40.75, 37.58, and 21.67 (metestrus); and 41.07, 37.38, and 21.67 (diestrus), respectively. In conclusion, dominant proportion of superficial cell that occurred in estrus period might be used as the base for determining optimal time for insemination. PMID:26977335

  17. Epithelial Cell Shedding and Barrier Function

    PubMed Central

    Williams, J. M.; Duckworth, C. A.; Burkitt, M. D.; Watson, A. J. M.; Campbell, B. J.

    2015-01-01

    The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed. PMID:25428410

  18. 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...

  19. Replication of cultured lung epithelial cells

    SciTech Connect

    Guzowski, D.; Bienkowski, R.

    1986-03-05

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to (/sup 3/H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems.

  20. Vaginal tumors

    MedlinePLUS

    Vaginal cancer; Cancer - vagina; Tumor - vaginal ... Most cancerous vaginal tumors occur when another cancer, such as cervical or endometrial cancer , spreads. This is called secondary vaginal cancer. Primary vaginal cancer is rare. Most primary vaginal cancers start ...

  1. What Is Vaginal Cancer?

    MedlinePLUS

    ... Cancer? There are several types of vaginal cancer. Squamous cell carcinoma About 70 of every 100 cases of vaginal cancer are squamous cell carcinomas . These cancers begin in the squamous cells that ...

  2. Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

    PubMed

    Goto, Yoshiyuki; Obata, Takashi; Kunisawa, Jun; Sato, Shintaro; Ivanov, Ivaylo I; Lamichhane, Aayam; Takeyama, Natsumi; Kamioka, Mariko; Sakamoto, Mitsuo; Matsuki, Takahiro; Setoyama, Hiromi; Imaoka, Akemi; Uematsu, Satoshi; Akira, Shizuo; Domino, Steven E; Kulig, Paulina; Becher, Burkhard; Renauld, Jean-Christophe; Sasakawa, Chihiro; Umesaki, Yoshinori; Benno, Yoshimi; Kiyono, Hiroshi

    2014-09-12

    Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation. PMID:25214634

  3. Innate lymphoid cells regulate intestinal epithelial cell glycosylation

    PubMed Central

    Goto, Yoshiyuki; Obata, Takashi; Kunisawa, Jun; Sato, Shintaro; Ivanov, Ivaylo I.; Lamichhane, Aayam; Takeyama, Natsumi; Kamioka, Mariko; Sakamoto, Mitsuo; Matsuki, Takahiro; Setoyama, Hiromi; Imaoka, Akemi; Uematsu, Satoshi; Akira, Shizuo; Domino, Steven E.; Kulig, Paulina; Becher, Burkhard; Renauld, Jean-Christophe; Sasakawa, Chihiro; Umesaki, Yoshinori; Benno, Yoshimi; Kiyono, Hiroshi

    2016-01-01

    Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host–microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria–dependent and –independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation. PMID:25214634

  4. Association of mesenchymal cells and immunoglobulins with differentiating epithelial cells

    PubMed Central

    Bukovsky, Antonin; Caudle, Michael R; Keenan, Jeffrey A; Upadhyaya, Nirmala B; Van Meter, Stuart E; Wimalasena, Jay; Elder, Robert F

    2001-01-01

    Background Mesenchymal-epithelial interactions play an important role in the physiology and pathology of epithelial tissues. Mesenchymal cells either associate with epithelium basement membrane [pericytes and perivascular monocyte-derived cells (MDC)] or reside within epithelium (MDC and T cells). Although intraepithelial mesenchymal cells were suggested to contribute to the epithelium physiology, their association with particular steps in differentiation of epithelial cells, interactions among themselves, and their fate remain unclear. We studied epitopes of mesenchymal cells and their products (immunoglobulins) in stratified epithelium of uterine ectocervix, which is one of the prototypes of complete cellular differentiation from stem into the aged cells. Results Perivascular CD14 primitive MDC associated with basal (stem) epithelial cells. Thy-1 pericytes of microvasculature secreted intercellular vesicles, which associated with Ki67 postmitotic epithelial cells expressing MHC class I. Intraepithelial T cells showed an association with veiled type MDC [dendritic cell (DC) precursors] among parabasal cells, and exhibited fragmentation after entering intermediate (mature) epithelial layers. Mature DC secreted CD68 and exhibited fragmentation after reaching mid intermediate layers. Binding of IgM was detected at the top of each layer: in the upper parabasal, upper intermediate, and most surface epithelial cells. IgG was confined to the entire superficial layer. Conclusions These data suggest that the phylogenetically and ontogenetically developed hierarchy of mesenchymal cells (MDC, pericytes, T cells) and immunoglobulins (IgM, IgG) accompanies differentiation of epithelial cells from immature into the mature and aged phenotype. Further studies of an involvement of mesenchymal cells in the regulation of tissue homeostasis may bring novel approaches to the prevention and therapy of tissue dysfunctions characterized by permanent tissue immaturity (muscular dystrophy) or accelerated aging (degenerative diseases). PMID:11439174

  5. Respiratory epithelial cells orchestrate pulmonary innate immunity

    PubMed Central

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

    The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and ‘instruct’ the professional immune system to protect the lungs from infection and injury. PMID:25521682

  6. The Human Airway Epithelial Basal Cell Transcriptome

    PubMed Central

    Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

    2011-01-01

    Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the human airway basal cell signature as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium. PMID:21572528

  7. The vaginal spindle cell epithelioma: a case report, review of the literature and discussion of potential histogenesis.

    PubMed

    Mahe, Etienne; Bishara, Mona; El Demellawy, Dina; DeNardi, Franco; Alowami, Salem

    2012-07-15

    The so-called mixed tumors occur in a variety of sites throughout the body. While most cases are encountered in the salivary glands, several cases have been described in the female genital tract. A variety of monikers have been applied to this lesion including "spindle cell epithelioma." As in other locations, the vaginal spindle cell epithelioma (VSE) consists of a proliferation of both epithelial and mesenchymal components. Based on our extensive review of the literature, we present the 53rd reported case of VSE. More significantly, we present the most up-to-date review of this lesion, including its immunohistochemical and electron microscopic features. We also review the theories pertaining to its histogenesis incorporating current embryologic data, which together suggest a Mllerian derivation. PMID:22703960

  8. EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.

    PubMed

    Kajita, Mihoko; Fujita, Yasuyuki

    2015-07-01

    During embryonic development or under certain pathological conditions, viable but suboptimal cells are often eliminated from the cellular society through a process termed cell competition. Cell competition was originally identified in Drosophila where cells with different properties compete for survival; 'loser' cells are eliminated from tissues and consequently 'winner' cells become dominant. Recent studies have shown that cell competition also occurs in mammals. While apoptotic cell death is the major fate for losers in Drosophila, outcompeted cells show more variable phenotypes in mammals, such as cell death-independent apical extrusion and cellular senescence. Molecular mechanisms underlying these processes have been recently revealed. Especially, in epithelial tissues, normal cells sense and actively eliminate the neighbouring transformed cells via cytoskeletal proteins by the process named epithelial defence against cancer (EDAC). Here, we introduce this newly emerging research field: cell competition in mammals. PMID:25991731

  9. Force mapping in epithelial cell migration

    NASA Astrophysics Data System (ADS)

    Du Roure, Olivia; Saez, Alexandre; Buguin, Axel; Austin, Robert H.; Chavrier, Philippe; Siberzan, Pascal; Ladoux, Benoit

    2005-02-01

    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 m) 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. cell mechanics | microfabrication | traction forces | multiple particle tracking

  10. Hyaluronan Rafts on Airway Epithelial Cells.

    PubMed

    Abbadi, Amina; Lauer, Mark; Swaidani, Shadi; Wang, Aimin; Hascall, Vincent

    2016-01-15

    Many cells, including murine airway epithelial cells, respond to a variety of inflammatory stimuli by synthesizing leukocyte-adhesive hyaluronan (HA) cables that remain attached to their cell surfaces. This study shows that air-liquid interface cultures of murine airway epithelial cells (AECs) also actively synthesize and release a majority of their HA onto their ciliated apical surfaces to form a heavy chain hyaluronan (HC-HA) matrix in the absence of inflammatory stimuli. These matrices do not resemble the rope-like HA cables but occur in distinct sheets or rafts that can capture and embed leukocytes from cell suspensions. The HC-HA modification involves the transfer of heavy chains from the inter-α-inhibitor (IαI) proteoglycan, which has two heavy chains (HC1 and HC2) on its chondroitin sulfate chain. The transesterification transfer of HCs from chondroitin sulfate to HA is mediated by tumor necrosis factor-induced gene 6 (TSG-6), which is up-regulated in inflammatory reactions. Because the AEC cultures do not have TSG-6 nor serum, the source of IαI, assays for HCs and TSG-6 were done. The results show that AECs synthesize TSG-6 and their own heavy chain donor (pre-IαI) with a single heavy chain 3 (HC3), which are also constitutively expressed by human renal proximal tubular epithelial cells. These leukocyte adhesive HC3-HA structures were also found in the bronchoalveolar lavage of naïve mice and were observed on their apical ciliated surfaces. Thus, these leukocyte-adhesive HA rafts are now identified as HC3-HA complexes that could be part of a host defense mechanism filling some important gaps in our current understanding of murine airway epithelial biology and secretions. PMID:26601955

  11. 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.

  12. Epithelial Cell Apoptosis in Recurrent Aphthous Ulcers.

    PubMed

    Al-Samadi, A; Drozd, A; Salem, A; Hietanen, J; Hyrinen-Immonen, R; Konttinen, Y T

    2015-07-01

    A recurrent aphthous ulcer (RAU) is a common inflammatory ulcerative lesion affecting oral mucosa. We studied the eventual apoptosis of epithelial cells from the point of view of ulcer and inflammation. RAU lesions and healthy mucosa samples were immunostained for caspase-3 and high-mobility group box 1 (HMGB1). DNA nicks were identified using TUNEL staining. We studied the effects of tumor necrosis factor ? (TNF?) and interferon ? (IFN?) on the toll-like receptor 2 and 4 (TLR2 and TLR4) expression of human oral SCC-25 keratinocytes. We also studied the effects of self-DNA, all-thiol-HMGB1, and disulfide-HMGB1 on epithelial cells, with or without IFN?. At the edge of RAU lesions, all epithelial cell layers were caspase-3(+), TUNEL(+), and HMGB-1(+) and had widened intercellular spaces. In contrast, healthy epithelial cells were negative for caspase-3 and TUNEL staining. HMGB1 was seen in only the basal cell layers, and the cells retained close cell-to-cell contacts. Self-DNA increased TNF-? mRNA (P = 0.02) in SCC-25 cells. Both TNF? and IFN? (P = 0.01) increased TLR2. Upon TNF? stimulation, SCC-25 cells lost their nuclear HMGB1 staining. HMGB1 did not increase IL-8, IL-6, or TNF-? mRNA in SCC-25 cells, which was unaffected by the presence of IFN?. We conclude that in healthy epithelium, the most superficial cells at the end of their life cycle are simply desquamated. In contrast, RAU is characterized by top-to-bottom apoptosis such that dead cells may slough off, leading to an ulcer. Because of a lack of scavenging anti-inflammatory macrophages, apoptotic cells probably undergo secondary necrosis releasing proinflammatory danger signals, which may contribute to the peripheral inflammatory halo. This is supported by self-DNA-induced TNF? synthesis. In contrast to TLR4- and TLR2-binding lipopolysaccharide used as a positive control, disulfide-HMGB1 did not stimulate proinflammatory cytokines. PMID:25861801

  13. Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice

    PubMed Central

    Pericolini, Eva; Gabrielli, Elena; Amacker, Mario; Kasper, Lydia; Roselletti, Elena; Luciano, Eugenio; Sabbatini, Samuele; Kaeser, Matthias; Moser, Christian; Hube, Bernhard; Vecchiarelli, Anna

    2015-01-01

    ABSTRACT Vaginal inflammation (vaginitis) is the most common disease caused by the human-pathogenic fungus Candida albicans. Secretory aspartyl proteinases (Sap) are major virulence traits of C.albicans that have been suggested to play a role in vaginitis. To dissect the mechanisms by which Sap play this role, Sap2, a dominantly expressed member of the Sap family and a putative constituent of an anti-Candida vaccine, was used. Injection of full-length Sap2 into the mouse vagina caused local neutrophil influx and accumulation of the inflammasome-dependent interleukin-1? (IL-1?) but not of inflammasome-independent tumor necrosis factor alpha. Sap2 could be replaced by other Sap, while no inflammation was induced by the vaccine antigen, the N-terminal-truncated, enzymatically inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library, inhibited or abolished the inflammatory response, provided the antibodies were able, like the Sap inhibitor Pepstatin A, to inhibit Sap enzyme activity. The same antibodies and Pepstatin A also inhibited neutrophil influx and cytokine production stimulated by C.albicans intravaginal injection, and a mutant strain lacking SAP1, SAP2, and SAP3 was unable to cause vaginal inflammation. Sap2 induced expression of activated caspase-1 in murine and human vaginal epithelial cells. Caspase-1 inhibition downregulated IL-1? and IL-18 production by vaginal epithelial cells, and blockade of the IL-1? receptor strongly reduced neutrophil influx. Overall, the data suggest that some Sap, particularly Sap2, are proinflammatory proteins in vivo and can mediate the inflammasome-dependent, acute inflammatory response of vaginal epithelial cells to C.albicans. These findings support the notion that vaccine-induced or passively administered anti-Sap antibodies could contribute to control vaginitis. PMID:26037125

  14. RAC1 overexpression promotes the proliferation, migration and epithelial-mesenchymal transition of lens epithelial cells

    PubMed Central

    Su, Jing; Li, Hong

    2015-01-01

    Cataract is a main cause of blindness worldwide. RAC1 has been reported to have a close relationship with the proliferation and migration of cells. However, the relationship between RAC1 and cataract is not yet clear. The proliferation and migration of lens epithelial cells are key factors in the formation of cataract as well as in the complication of cataract surgery. In this study, the effect of RAC1 overexpression on the proliferation and migration of lens epithelial cells was explored. Results showed that RAC1 overexpression promoted the proliferation of lens epithelial cells and increased the protein level of proliferating cell nuclear antigen. RAC1 overexpression also promoted migration and invasion of lens epithelial cells and had an influence on the epithelial-mesenchymal transition process. These results indicate that RAC1 may become a therapeutic target of cataract and inhibition of RAC1 may become a promising way for the therapy of cataract. PMID:26617787

  15. Preservative cytotoxicity to cultured corneal epithelial cells.

    PubMed

    Neville, R; Dennis, P; Sens, D; Crouch, R

    1986-05-01

    Cultured human and rat corneal epithelial cells with 51Cr incorporated were used as a model to test the cytolytic action of four common preservatives. Benzalkonium chloride, chlorohexidine and thimerosol were all found to lyse greater than 40% cells when incubated for fifteen minutes at concentrations in clinical use in topical ophthalmic medications. Chlorobutanol is the only preservative tested which has a low level of cytotoxicity (10%) and which, under these conditions, can be considered a safe preservative using cytolytic activity as the means of criteria. PMID:3720343

  16. Immortalization of bovine pancreatic duct epithelial cells.

    PubMed

    Marino, L R; Cotton, C U

    1996-04-01

    Pancreatic duct cell lines have been isolated from a number of animal and human tumors, but none appear to express ion transport properties expected for differentiated pancreatic duct epithelial cells. We sought to generate an immortalized ductal cell line from well-differentiated primary cultures of bovine pancreatic duct epithelium. Epithelial cells from the main duct of the bovine pancreas were isolated and immortalized by transfection with a DNA construct encoding simian virus 40 large T antigen. A single clone (BPD1) survived negative selection and was maintained in culture for > 100 passages over 2 yr. The cells grow readily in culture as monolayers and express several properties characteristic of differentiated pancreatic ductal epithelium. The cells do not appear to form a functional tight junction complex, since the transepithelial resistance of the monolayer cultures grown on a permeable support is < 10 omega.cm2. Northern blot analysis revealed that the cells continue to express simian virus 40 large T antigen and contain significant levels of mRNA for proteins thought to be important in transepithelial bicarbonate secretion [carbonic anhydrase II, Cl-/HCO3- exchanger, Na+/H+ exchanger, and cystic fibrosis transmembrane conductance regulator (CFTR)]. In vivo pancreatic ductal secretion is stimulated by the peptide hormone secretin. The secretin receptor is expressed and functionally coupled to adenylate cyclase in the immortalized cells, since secretin caused a dose-dependent accumulation of adenosine 3'5'-cyclic monophosphate (cAMP; approximately 20-fold increase over basal levels) with a mean effective concentration of 15 nM. Elevation of intracellular cAMP by exposure of the cells to forskolin (10 microM) or secretin (0.1 microM) increase plasma membrane Cl- permeability, most likely mediated by activation of CFTR. The results of these studies demonstrate that the pancreatic duct cell line (BPD1) retains several properties exhibited by the secretory epithelial cells that line the pancreatic ductal tree. This cell line should prove useful for studies of expression, function, and regulation of pancreatic duct cell proteins. PMID:8928798

  17. 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

  18. Hippo signaling in epithelial stem cells.

    PubMed

    Yin, Meng-Xin; Zhang, Lei

    2015-01-01

    Over the past decade, discoveries on Hippo signaling have revealed a complex signaling network integrating various signaling pathways to modulate tissue homeostasis, organ size control, tissue repair, and regeneration. Malfunction of the Hippo pathway is associated with tumor and cancer development. Moreover, Hippo signaling has been proposed to act in numerous stem cells in a variety of organisms. Recently, more attention has been paid to define the functions of the Hippo pathway in tissue-specific stem cells, which have great potential to be used in cell-based therapies. Here we provide an overview of its roles in regulating stem cells in epithelial tissues and its potential implications in related cancers. PMID:25476205

  19. 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, ?SMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of ?SMA, Smad3 and Smad4, TGF? signaling intermediates, accumulated in the nucleus and Snail, a TGF? 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 TGF? signaling. PMID:24095903

  20. Ouabain modulates ciliogenesis in epithelial cells.

    PubMed

    Larre, Isabel; Castillo, Aida; Flores-Maldonado, Catalina; Contreras, Ruben G; Galvan, Ivan; Muñoz-Estrada, Jesus; Cereijido, Marcelino

    2011-12-20

    The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na(+) and H(2)O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na(+),K(+)-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life. PMID:22143774

  1. Vaginal Infections

    MedlinePLUS

    ... Home Body Your reproductive health Vaginal infections Vaginal infections Help for infections If you have pain, itching, ... and how to prevent them. Types of vaginal infections top Two common vaginal infections are bacterial vaginosis ...

  2. Establishment of Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium.

    PubMed

    Nam, Hyun; Kim, Ji-Hye; Kim, Jae-Won; Seo, Byoung-Moo; Park, Joo-Cheol; Kim, Jung-Wook; Lee, Gene

    2014-07-01

    Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-?1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth. PMID:25081036

  3. 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.

  4. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz; Rockx, Barry

    2013-03-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1?, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  5. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  6. NK cell responses to simian immunodeficiency virus vaginal exposure in naive and vaccinated rhesus macaques.

    PubMed

    Shang, Liang; Smith, Anthony J; Duan, Lijie; Perkey, Katherine E; Qu, Lucy; Wietgrefe, Stephen; Zupancic, Mary; Southern, Peter J; Masek-Hammerman, Katherine; Reeves, R Keith; Johnson, R Paul; Haase, Ashley T

    2014-07-01

    NK cell responses to HIV/SIV infection have been well studied in acute and chronic infected patients/monkeys, but little is known about NK cells during viral transmission, particularly in mucosal tissues. In this article, we report a systematic study of NK cell responses to high-dose vaginal exposure to SIVmac251 in the rhesus macaque female reproductive tract (FRT). Small numbers of NK cells were recruited into the FRT mucosa following vaginal inoculation. The influx of mucosal NK cells preceded local virus replication and peaked at 1 wk and, thus, was in an appropriate time frame to control an expanding population of infected cells at the portal of entry. However, NK cells were greatly outnumbered by recruited target cells that fuel local virus expansion and were spatially dissociated from SIV RNA+ cells at the major site of expansion of infected founder populations in the transition zone and adjoining endocervix. The number of NK cells in the FRT mucosa decreased rapidly in the second week, while the number of SIV RNA+ cells in the FRT reached its peak. Mucosal NK cells produced IFN-? and MIP-1?/CCL3 but lacked several markers of activation and cytotoxicity, and this was correlated with inoculum-induced upregulation of the inhibitory ligand HLA-E and downregulation of the activating receptor CD122/IL-2R?. Examination of SIV?nef-vaccinated monkeys suggested that recruitment of NK cells to the genital mucosa was not involved in vaccine-induced protection from vaginal challenge. In summary, our results suggest that NK cells play, at most, a limited role in defenses in the FRT against vaginal challenge. PMID:24899503

  7. Dedifferentiation of committed epithelial cells into stem cells in vivo.

    PubMed

    Tata, Purushothama Rao; Mou, Hongmei; Pardo-Saganta, Ana; Zhao, Rui; Prabhu, Mythili; Law, Brandon M; Vinarsky, Vladimir; Cho, Josalyn L; Breton, Sylvie; Sahay, Amar; Medoff, Benjamin D; Rajagopal, Jayaraj

    2013-11-14

    Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states, notably cancer. PMID:24196716

  8. Dedifferentiation of committed epithelial cells into stem cells in vivo

    PubMed Central

    Tata, Purushothama Rao; Mou, Hongmei; Pardo-Saganta, Ana; Zhao, Rui; Prabhu, Mythili; Prabhu, Mythili; Law, Brandon M.; Vinarsky, Vladimir; Cho, Josalyn L.; Breton, Sylvie; Sahay, Amar; Medoff, Benjamin D.; Rajagopal, Jayaraj

    2014-01-01

    Summary Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes, and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. We now present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. Following the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts to repair epithelial injury. Indeed, single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. In contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate was inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may play a more general role in the regeneration of many tissues and in multiple disease states, notably cancer. PMID:24196716

  9. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

    PubMed Central

    Celi-Terrassa, Toni; Meca-Corts, scar; Mateo, Francesca; Martnez de Paz, Alexia; Rubio, Nuria; Arnal-Estap, Anna; Ell, Brian J.; Bermudo, Raquel; Daz, Alba; Guerra-Rebollo, Marta; Lozano, Juan Jos; Estars, Conchi; Ulloa, Catalina; ?lvarez-Simn, Daniel; Mil, Jordi; Vilella, Ramn; Paciucci, Rosanna; Martnez-Balbs, Marian; Garca de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jernimo; Fernndez, Pedro L.; Thomson, Timothy M.

    2012-01-01

    Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

  10. 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 cells participate in angiotensin II-mediated control of the glomerular filtration barrier.

  11. Goat uterine epithelial cells are susceptible to infection with Caprine Arthritis Encephalitis Virus (CAEV) in vivo

    PubMed Central

    2012-01-01

    The aim of this study was to determine, using immunofluorescence and in situ hybridization, whether CAEV is capable of infecting goat uterine epithelial cells in vivo. Five CAEV seropositive goats confirmed as infected using double nested polymerase chain reaction (dnPCR) on leucocytes and on vaginal secretions were used as CAEV positive goats. Five CAEV-free goats were used as controls. Samples from the uterine horn were prepared for dnPCR, in situ hybridization, and immunofluorescence. The results from dnPCR confirmed the presence of CAEV proviral DNA in the uterine horn samples of infected goats whereas no CAEV proviral DNA was detected in samples taken from the uninfected control goats. The in situ hybridization probe was complementary to part of the CAEV gag gene and confirmed the presence of CAEV nucleic acids in uterine samples. The positively staining cells were seen concentrated in the mucosa of the lamina propria of uterine sections. Finally, laser confocal analysis of double p28/cytokeratin immunolabelled transverse sections of CAEV infected goat uterus, demonstrated that the virus was localized in glandular and epithelial cells. This study clearly demonstrates that goat uterine epithelial cells are susceptible to CAEV infection in vivo. This finding could help to further our understanding of the epidemiology of CAEV, and in particular the possibility of vertical transmission. PMID:22276529

  12. 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

  13. Uranium induces apoptosis in lung epithelial cells

    PubMed Central

    Periyakaruppan, Adaikkappan; Sarkar, Shubhashish; Ravichandran, Prabakaran; Sadanandan, Bindu; Sharma, Chidananda S.; Ramesh, Vani; Hall, Joseph C.; Thomas, Renard; Wilson, Bobby L.

    2009-01-01

    Uranium is a naturally occurring radioactive material present everywhere in the environment. It is toxic because of its chemical or radioactive properties. Uranium enters environment mainly from mines and industry and cause threat to human health by accumulating in lungs as a result of inhalation. In our previous study, we have shown the effectiveness of antioxidant system response to the oxidative stress induced by uranyl acetate (UA) in rat lung epithelial (LE) cells. As part of our continuing studies; here, we investigated the mechanism underlying when LE cells are exposed to different concentration of UA. Oxidative stress may lead to apoptotic signaling pathways. LE cells treated with 0.25, 0.5 and 1 mM of UA results in dose and time-dependent increase in activity of both caspases-3 and -8. Increase in the concentration of cytochrome-c oxidase in cytosol was seen in LE cells treated with 1 mM UA as a result of mitochondria membrane permeability. The cyto-chrome-c leakage may trigger the apoptotic pathway. TUNEL assay performed in LE cells treated with 1 mM of UA showed significant incorporation of dNTPs in the nucleus after 24 h. In the presence of the caspase inhibitors, we observed the significant decrease in the activity of caspases-8 and -3 in 0.5 and 1 mM UA-treated LE cells. PMID:19096828

  14. Multi-functionality and plasticity characterize epithelial cells in Hydra.

    PubMed

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  15. Multi-functionality and plasticity characterize epithelial cells in Hydra

    PubMed Central

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  16. 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. PMID:26778835

  17. Observing planar cell polarity in multiciliated mouse airway epithelial cells

    PubMed Central

    Vladar, Eszter K.; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D.

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  18. Observing planar cell polarity in multiciliated mouse airway epithelial cells.

    PubMed

    Vladar, Eszter K; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  19. Novel neurotrophic factor secreted by amniotic epithelial cells.

    PubMed

    Venkatachalam, Sankar; Palaniappan, Tamilselvi; Jayapal, Prem Kumar; Neelamegan, Sridharan; Rajan, Sridhar Skylab; Muthiah, Vijaya Prakash Krishnan

    2009-08-01

    By virtue of expressions of glial and neural surface markers and capability of neurotransmitter metabolism, amniotic epithelial cells are considered as candidate cell type for transplantation strategies to treat neurological disorders. Previously, we have reported neurotrophism exhibited by human amniotic epithelial cells when transplanted after spinal cord injury in bonnet monkeys. Amniotic epithelial cells were believed to secrete an "Epidermal Growth Factor (EGF)-like" factor and exact identification was not made. At this juncture, through the present study it was found that, chicken neural retinal cells when grown alone failed to survive and contrarily when either co-cultured with chicken amniotic epithelial cells/cultured in amniotic epithelial cell conditioned medium not only survived but also showed extensive differentiation. Fibroblast Growth Factor-2 (FGF-2) plays a critical role in retinal development especially in chicken neural retinal development. However, immunoassay using western blot did not revealed the presence of any already known isoforms of FGF-2 in the medium. It is interesting to note that while factor secreted by amniotic epithelial cells resembles EGF and/or FGF-2 in its biological action, known isoforms of them were not detected. Considering the biological closeness between EGF and FGF-2, results indicate the possibility of a novel isoform of these growth factors secreted by amniotic epithelial cells. Further studies will establish the nature of this novel factor which will enhance the application of this interesting cell type for neural transplantations. PMID:19886035

  20. Vaginal Microbiota and Sexually Transmitted Infections That May Influence Transmission of Cell-Associated HIV

    PubMed Central

    Cone, Richard A.

    2014-01-01

    Vaginal microbiota and sexually transmitted infections (STIs) are likely to influence the transmission of cell-associated human immunodeficiency virus (HIV). Lactic acid produced by Lactobacillus-dominated microbiota (Nugent score 03) will likely inhibit transmission, especially female-to-male transmission. In contrast, polymicrobial microbiota (Nugent score 410), community state types IV-A and IV-B, and STIs will likely increase transmission of cell-associated HIV. PMID:25414415

  1. Recycling of galectin-3 in epithelial cells.

    PubMed

    Hönig, Ellena; Schneider, Katharina; Jacob, Ralf

    2015-01-01

    Galectins, a family of β-galactoside binding proteins, do not possess a signalling sequence to enter the endoplasmic reticulum as a starting point for the classical secretory pathway. They use a so-called unconventional secretion mechanism for translocation across the plasma membrane and/or into the lumen of transport vesicles. The β-galactoside binding protein galectin-3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium. The lectin re-enters the cell by non-clathrin mediated endocytosis and passages through endosomal organelles. This internalized galectin-3 plays an important role in apical protein trafficking by directing the subcellular targeting of apical glycoproteins via oligomerization into high molecular weight clusters, a process that can be fine-tuned by changes in the environmental pH. Following release at the apical plasma membrane, the lectin can reenter the cell for another round of recycling and apical protein sorting. This review will briefly address galectin-3-functions in epithelia and focus on distinct phases in apical recycling of the lectin. PMID:26059399

  2. Extensive podocyte loss triggers a rapid parietal epithelial cell response.

    PubMed

    Hakroush, Samy; Cebulla, Angelika; Schaldecker, Thomas; Behr, Daniel; Mundel, Peter; Weins, Astrid

    2014-05-01

    Damage to podocytes is a central pathomechanism of proteinuric kidney disease. However, it is not fully understood how podocyte injury evolves to progressive glomerulopathies such as FSGS or collapsing glomerulopathy. In particular, the role of parietal epithelial cells remains controversial. Here, we show that adriamycin induces DNA damage and podocyte lysis in mice without evidence of autophagy, endoplasmic reticulum stress, or necroptosis. After extensive podocyte loss, activated parietal cells mediated tuft re-epithelialization by two distinct mechanisms. In the majority of glomeruli, vacuolized parietal epithelial cells attached to denuded glomerular basement membrane and, occasionally, disengaged from the parietal basement membrane. Less frequently, parietal epithelial cells covered the denuded visceral basement membrane via formation of proliferative pseudocrescents. Notably, "visceralized" parietal epithelial cells did not express vascular endothelial growth factor but upregulated hypoxia-inducible factor 1 expression. The presence of visceralized parietal epithelial cells in sclerosing and collapsing lesions in a kidney biopsy from a patient with diabetes underscores the human relevance of our findings. In conclusion, repopulation of the glomerular tuft by parietal cells may represent a compensatory response to extensive podocyte loss. Our results suggest, however, that visceralized parietal epithelial cells cannot induce revascularization of the hyalinized tuft, resulting in hypoxic cell death and irreversible destruction of the glomerulus. PMID:24335975

  3. Foxp3+ Regulatory T Cells Promote Lung Epithelial Proliferation

    PubMed Central

    Mock, Jason R.; Garibaldi, Brian T.; Aggarwal, Neil R.; Jenkins, John; Limjunyawong, Nathachit; Singer, Benjamin D.; Chau, Eric; Rabold, Richard; Files, Daniel C.; Sidhaye, Venkataramana; Mitzner, Wayne; Wagner, Elizabeth M.; King, Landon S.; DAlessio, Franco R.

    2014-01-01

    Acute Respiratory Distress Syndrome (ARDS) causes significant morbidity and mortality each year. There is a paucity of information regarding the mechanisms necessary for ARDS resolution. Foxp3+ regulatory T cells (Tregs) have been shown to be an important determinant of resolution in an experimental model of lung injury. We demonstrate that intratracheal delivery of endotoxin (LPS) elicits alveolar epithelial damage from which the epithelium undergoes proliferation and repair. Epithelial proliferation coincided with an increase in Foxp3+ Treg cells in the lung during the course of resolution. To dissect the role that Foxp3+ Treg cells exert on epithelial proliferation, we depleted Foxp3+ Treg cells which led to decreased alveolar epithelial proliferation and delayed lung injury recovery. Furthermore, antibody-mediated blockade of CD103, an integrin, which binds to epithelial expressed E-cadherin decreased Foxp3+ Treg numbers and decreased rates of epithelial proliferation after injury. In a non-inflammatory model of regenerative alveologenesis, left lung pneumonectomy (PNX), we found that Foxp3+ Treg cells enhanced epithelial proliferation. Moreover, Foxp3+ Treg cells co-cultured with primary type II alveolar cells (AT2) directly increased AT2 cell proliferation in a CD103-dependent manner. These studies provide evidence of a new and integral role for Foxp3+ Treg cells in repair of the lung epithelium. PMID:24850425

  4. Cytomatrix synthesis in MDCK epithelial cells

    SciTech Connect

    Mitchell, J.J.; Low, R.B.; Woodcock-Mitchell, J.L. )

    1990-06-01

    Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak, was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with (14C)leucine over several days and then pulse-labeled for 4 hours with (3H)leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form.

  5. Effect of Rabbit Epididymal Antimicrobial Peptide, REHb?P, on LPS-Induced Proinflammatory Cytokine Responses in Human Vaginal Cells In Vitro

    PubMed Central

    Reddy, K. V. R.; Sukanya, D.; Patgaonkar, M. S.; Selvaakumar, C.

    2012-01-01

    Antimicrobial peptides (AMP's) protect epithelial surfaces including epididymis against pathogens and play a key role in orchestrating various defensive responses. Recently, we have identified one such AMP, rabbit epididymal hemoglobin-? subuit (REHb?P) from the epididymal fluid of rabbit, Oryctologus cuniculus. The demonstration of a protective role of REHb?P in epididymal epithelial cells (EPEC's) led us to investigate: (1) the identification of LPS interactive domain in REHb?P, and (2) whether the REHb?P of rabbit origin mediates vaginal cellular immune responses of another species (human). HeLa-S3, human vaginal epithelial cells (hVECs) were exposed to LPS or the LPS-stimulated cells treated with REHb?P or neutral peptide, nREHb?P. Effect of LPS and cytokines (IL-6 and IL-1?) and chemokines (IL-8, MCP-1) levels was determined in the culture supernatants. In response to the LPS, hVECs synthesized these mediators and the levels were significantly higher than controls. This enhancing effect was ameliorated when the LPS-induced hVECs were treated with REHb?P. Similar results were obtained on NF-?B protein and hBD-1 mRNA expression. Confocal microscopy studies revealed that REHb?P attenuated the LPS-induced internalization of E. coli by macrophages. The chemotaxis studies performed using Boyden chamber Transwell assay, which showed elevated migration of U937 cells when the supernatants of LPS-induced hVECs were used, and the effect was inhibited by REHb?P. REHb?P was found to be localized on the acrosome of rabbit spermatozoa, suggesting its role in sperm protection beside sperm function. In conclusion, REHb?P may have the potential to develop as a therapeutic agent for reproductive tract infections (RTI's). PMID:22505946

  6. Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration.

    PubMed

    Le Guen, Ludovic; Marchal, Stphane; Faure, Sandrine; de Santa Barbara, Pascal

    2015-10-01

    The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration. PMID:26126787

  7. Vaginal cysts

    MedlinePLUS

    ... with air, fluid, pus, or other material. A vaginal cyst occurs on or under the vaginal lining. ... There are several types of vaginal cysts. Vaginal inclusion cysts are the most common. These may form as a result of injury to the vaginal walls during ...

  8. Quantitative Assessment of Cytosolic Salmonella in Epithelial Cells

    PubMed Central

    Knodler, Leigh A.; Nair, Vinod; Steele-Mortimer, Olivia

    2014-01-01

    Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol. PMID:24400108

  9. Limbal epithelial stem cell identification using immunoblotting analysis.

    PubMed

    Wright, Bernice; Connon, Che J

    2013-01-01

    The unambiguous identification of limbal epithelial stem cells is currently a major challenge in corneal stem cell biology. Specific molecular markers which characterize these cells are lacking. At present, the best strategy for identification of limbal epithelial stem cells is to investigate a variety of putative markers for these cells in a differentiated (cytokeratin (CK) 3: CK3, integrin ?6), undifferentiated (CK14), and naive state (?Np63?, ATP-binding cassette subfamily G member 2 (ABCG2), integrin ?9, Notch-1), alongside functional assays which indicate their stemness. The focus of this chapter is to highlight advances in the Western blotting technique for quantitative assessment of corneal epithelial cell markers, and the use of this technique for investigation of a range of different protein markers which identify limbal epithelial stem cells. PMID:23690007

  10. Gremlin Activates the Smad Pathway Linked to Epithelial Mesenchymal Transdifferentiation in Cultured Tubular Epithelial Cells

    PubMed Central

    Rodrigues-Diez, Raquel; Rodrigues-Diez, Ral R.; Lavoz, Carolina; Carvajal, Gisselle; Droguett, Alejandra; Garcia-Redondo, Ana B.; Rodriguez, Isabel; Ortiz, Alberto; Egido, Jess; Mezzano, Sergio; Ruiz-Ortega, Marta

    2014-01-01

    Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-? (TGF-?). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-? mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF-?, by a neutralizing antibody against active TGF-?, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-? independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-? production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-? neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-?. PMID:24949470

  11. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjrn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

  12. Intrinsic epithelial cells repair the kidney after injury.

    PubMed

    Humphreys, Benjamin D; Valerius, M Todd; Kobayashi, Akio; Mugford, Joshua W; Soeung, Savuth; Duffield, Jeremy S; McMahon, Andrew P; Bonventre, Joseph V

    2008-03-01

    Understanding the mechanisms of nephron repair is critical for the design of new therapeutic approaches to treat kidney disease. The kidney can repair after even a severe insult, but whether adult stem or progenitor cells contribute to epithelial renewal after injury and the cellular origin of regenerating cells remain controversial. Using genetic fate-mapping techniques, we generated transgenic mice in which 94%-95% of tubular epithelial cells, but no interstitial cells, were labeled with either beta-galactosidase (lacZ) or red fluorescent protein (RFP). Two days after ischemia-reperfusion injury (IRI), 50.5% of outer medullary epithelial cells coexpress Ki67 and RFP, indicating that differentiated epithelial cells that survived injury undergo proliferative expansion. After repair was complete, 66.9% of epithelial cells had incorporated BrdU, compared to only 3.5% of cells in the uninjured kidney. Despite this extensive cell proliferation, no dilution of either cell-fate marker was observed after repair. These results indicate that regeneration by surviving tubular epithelial cells is the predominant mechanism of repair after ischemic tubular injury in the adult mammalian kidney. PMID:18371453

  13. Maintenance and Distribution of Epithelial Stem/Progenitor Cells after Corneal Reconstruction Using Oral Mucosal Epithelial Cell Sheets

    PubMed Central

    Soma, Takeshi; Hayashi, Ryuhei; Sugiyama, Hiroaki; Tsujikawa, Motokazu; Kanayama, Shintaro; Oie, Yoshinori; Nishida, Kohji

    2014-01-01

    We assessed the maintenance and distribution of epithelial stem/progenitor cells after corneal reconstruction using tissue-engineered oral mucosal cell sheets in a rat model. Oral mucosal biopsy specimens were excised from green fluorescent protein (GFP) rats and enzymatically treated with Dispase II. These cells were cultured on inserts with mitomycin C-treated NIH/3T3 cells, and the resulting cell sheets were harvested. These tissue-engineered cell sheets from GFP rats were transplanted onto the eyes of a nude rat limbal stem cell deficiency model. Eight weeks after surgery, ocular surfaces were completely covered by the epithelium with GFP-positive cells. Transplanted corneas expressed p63 in the basal layers and K14 in all epithelial layers. Epithelial cells harvested from the central and peripheral areas of reconstructed corneas were isolated for a colony-forming assay, which showed that the colony-forming efficiency of the peripheral epithelial cells was significantly higher than that of the central epithelial cells 8 weeks after corneal reconstruction. Thus, in this rat model, the peripheral cornea could maintain more stem/progenitor cells than the central cornea after corneal reconstruction using oral mucosal epithelial cell sheets. PMID:25343456

  14. Vaginal Diseases

    MedlinePLUS

    Vaginal problems are some of the most common reasons women go to the doctor. They may have ... the problem is vaginitis, an inflammation of the vagina. The main symptom is smelly vaginal discharge, but ...

  15. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  16. Endoplasmic reticulum protein 29 regulates epithelial cell integrity during the mesenchymal-epithelial transition in breast cancer cells.

    PubMed

    Bambang, I F; Lee, Y K; Richardson, D R; Zhang, D

    2013-03-01

    The epithelial-mesenchymal transition (EMT) correlates with disruption of cell-cell adhesion, loss of cell polarity and development of epithelial cell malignancy. Identifying novel molecules that inhibit EMT has profound potential for developing mechanism-based therapeutics. We previously demonstrated that the endoplasmic reticulum protein 29 (ERp29) is a novel factor that can drive mesenchymal-epithelial transition (MET) and induce cell growth arrest in MDA-MB-231 cells. Here, we show that ERp29 is an important molecule in establishing epithelial cell integrity during the MET. We demonstrate that ERp29 regulates MET in a cell context-dependent manner. ERp29 overexpression induced a complete MET in mesenchymal MDA-MB-231 cells through downregulating the expression of transcriptional repressors (for example, Slug, Snai1, ZEB2 and Twist) of E-cadherin. In contrast, overexpression of ERp29 induces incomplete MET in basal-like BT549 cells in which the expression of EMT-related markers (for example, vimentin; cytokeratin 19 (CK19) and E-cadherin) and the transcriptional repressors of E-cadherin were not altered. However, ERp29 overexpression in both cell-types resulted in loss of filamentous stress fibers, formation of cortical actin and restoration of an epithelial phenotype. Mechanistic studies revealed that overexpression of ERp29 in both cell-types upregulated the expression of TJ proteins (zonula-occludens-1 (ZO-1) and occludin) and the core apical-basal polarity proteins (Par3 and Scribble) at the membrane to enhance cell-cell contact and cell polarization. Knockdown of ERp29 in the epithelial MCF-7 cells decreased the expression of these proteins, leading to the disruption of cell-cell adhesion. Taken together, ERp29 is a novel molecule that regulates MET and epithelial cell integrity in breast cancer cells. PMID:22543584

  17. Genetics and epithelial cell dysfunction in cystic fibrosis

    SciTech Connect

    Riordan, J.R.; Buchwald, M.

    1987-01-01

    This book examines the advances being made in the study of the physiology, cell biology, and molecular genetics of cystic fibrosis. Emphasis is placed on various areas of research that involve epithelial cells (e.g., the CF-specific phenotypes exhibited by epithelial cells, abnormalities in epithelium ion transport, chloride channel regulation in CF epithelial.) Coverage is presented on the current status of CF, including data on the incidence of the disease, its mode of inheritance, chromosomal localization, genetic heterogeneity, and screening and management.

  18. Alignment of cell division axes in directed epithelial cell migration

    NASA Astrophysics Data System (ADS)

    Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rdler, Joachim; Elgeti, Jens

    2014-11-01

    Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

  19. 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

  20. Emergence of an Apical Epithelial Cell Surface InVivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Mat; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, wecombine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium invivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging celland mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

  1. SWCNTs induced autophagic cell death in human bronchial epithelial cells.

    PubMed

    Park, Eun-Jung; Zahari, Nur Elida M; Lee, Eun-Woo; Song, Jaewhan; Lee, Jae-Hyeok; Cho, Myung-Haing; Kim, Jae-Ho

    2014-04-01

    Carbon nanotubes are being actively introduced in electronics, computer science, aerospace, and other industries. Thus, the urgent need for toxicological studies on CNTs is mounting. In this study, we investigated the alterations in cellular response with morphological changes induced by single-walled carbon nanotubes (SWCNTs) in BEAS-2B cells, a human bronchial epithelial cell line. At 24h after exposure, SWCNTs rapidly decreased ATP production and cell viability as well a slight increase in the number of cells in the subG1 and G1 phases. In addition, SWCNTs increased the expression of superoxide dismutase (SOD)-1, but not SOD-2, and the number of cells generating ROS. The concentration of Cu and Zn ions also increased in a dose-dependent manner in cells exposed to SWCNTs. SWCNTs significantly enhanced the release of nitric oxide, interleukin (IL)-6, and IL-8 and up-regulated the expression of chemokine- and cytokine-related genes. Furthermore, the levels of autophagy-related genes, especially the DRAM1 gene, and the autophagosome formation-related proteins, were clearly up-regulated together with an increase of autophagosome-like vacuoles. Based on these results, we suggest that SWCNTs induce autophagic cell death through mitochondrial dysfunction and cytosolic damage in human bronchial epithelial cells. PMID:24389112

  2. Modulation of cell surface-associated mannoprotein antigen expression in experimental candidal vaginitis.

    PubMed Central

    De Bernardis, F; Molinari, A; Boccanera, M; Stringaro, A; Robert, R; Senet, J M; Arancia, G; Cassone, A

    1994-01-01

    The monoclonal antibody (MAb) AF1 recognizes an oligosaccharide epitope present on highly immunogenic and immunomodulatory mannoproteins (MP) of Candida albicans. The expression of this epitope (AF1-MP) during experimental candidal vaginitis was studied in two strains of C. albicans (3153 and CA-2) which were equally vaginopathic but differed in the mode of hypha formation in the vagina. In both strains, immunofluorescence of vaginal samples, taken 1 h after challenge, revealed an intense, MAb AF1-specific labelling of the yeast cells. This labelling was very scarce in fungal cells taken at 24 h and on subsequent days during the development of filamentous forms. Electron-microscopic gold immunolabelling observations showed that molecules carrying AF1-MP spanned the entire cell wall in the initial yeast cells but were absent on the cell surface and in the outermost, capsular layer of the cell wall of the germ tubes and filamentous forms. In both strains, at any time and for any form of intravaginal growth, AF1-MP was clearly expressed in the cytoplasm and cytoplasmic vesicles, and was fully incorporated into the inner layers of the cell wall. As seen by immunofluorescence, the vaginal fluid from C. albicans-infected rats did not hinder the expression of AF1-MP on the yeast cells surface in vitro. In electron-microscopic gold immunolabelling, a hypha-specific MAb (3D9) labelled the surface of the hyphal but not of the yeast cells of C. albicans harvested from rat vagina. Overall, these data strongly suggest that cell surface expression of MP antigen is modulated during intravaginal growth and morphogenesis of C. albicans. Images PMID:7507895

  3. Diversity of Epithelial Stem Cell Types in Adult Lung

    PubMed Central

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  4. Characterization of primary cultures of adult human epididymis epithelial cells

    PubMed Central

    Leir, Shih-Hsing; Browne, James A.; Eggener, Scott E.; Harris, Ann

    2014-01-01

    Objective To establish cultures of epithelial cells from all regions of the human epididymis to provide reagents for molecular approaches to functional studies of this epithelium. Design Experimental laboratory study. Setting University research institute. Patient(s) Epididymis from seven patients undergoing orchiectomy for suspected testicular cancer without epididymal involvement. Intervention(s) Human epididymis epithelial cells harvested from adult epididymis tissue. Main Outcome Measure(s) Establishment of a robust culture protocol for adult human epididymal epithelial cells. Result(s) Cultures of caput, corpus, and cauda epithelial cells were established from epididymis tissue of seven donors. Cells were passaged up to eight times and maintained differentiation markers. They were also cryopreserved and recovered successfully. Androgen receptor, clusterin, and cysteine-rich secretory protein 1 were expressed in cultured cells, as shown by means of immunofluorescence, Western blot, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The distribution of other epididymis markers was also shown by means of qRT-PCR. Cultures developed transepithelial resistance (TER), which was androgen responsive in the caput but androgen insensitive in the corpus and cauda, where unstimulated TER values were much higher. Conclusion(s) The results demonstrate a robust in vitro culture system for differentiated epithelial cell types in the caput, corpus, and cauda of the human epididymis. These cells will be a valuable resource for molecular analysis of epididymis epithelial function, which has a pivotal role in male fertility. PMID:25542823

  5. HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells

    PubMed Central

    Tugizov, Sharof M.; Herrera, Rossana; Veluppillai, Piri; Greenspan, Deborah; Soros, Vanessa; Greene, Warner C.; Levy, Jay A.; Palefsky, Joel M.

    2010-01-01

    Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells – and not those that passed through the adult cells – remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission. PMID:21056450

  6. Stochastic Terminal Dynamics in Epithelial Cell Intercalation

    NASA Astrophysics Data System (ADS)

    Eule, Stephan; Metzger, Jakob; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Grosshans, Joerg; Wolf, Fred

    2015-03-01

    We found that the constriction of epithelial cell contacts during intercalation in germ band extension in Drosophila embryos follows intriguingly simple quantitative laws. The mean contact length < L > follows < L > (t) ~(T - t) α , where T is the finite collapse time; the time dependent variance of contact length is proportional to the square of the mean; finally the time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations suggest that the dynamics of contact collapse can be captured by a stochastic differential equation analytically tractable in small noise approximation. Here, we present such a model, providing an effective description of the non-equilibrium statistical mechanics of contact collapse. All model parameters are fixed by measurements of time dependent mean and variance of contact lengths. The model predicts the contact length covariance function that we obtain in closed form. The contact length covariance function closely matches experimental observations suggesting that the model well captures the dynamics of contact collapse.

  7. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    SciTech Connect

    Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot; Egan, Jim J.; Doran, Peter P.

    2009-07-01

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  8. 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. PMID:20400167

  9. Cell volume regulation in epithelial physiology and cancer

    PubMed Central

    Pedersen, Stine F.; Hoffmann, Else K.; Novak, Ivana

    2013-01-01

    The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered expression of ion transporters and channels is now recognized as one of the hallmarks of cancer, it is timely to consider this especially for epithelia. Epithelial cells are highly proliferative and epithelial cancers, carcinomas, account for about 90% of all cancers. In this review we will focus on ion transporters and channels with key physiological functions in epithelia and known roles in the development of cancer in these tissues. Their roles in cell survival, cell cycle progression, and development of drug resistance in epithelial cancers will be discussed. PMID:24009588

  10. Regulated Mucin Secretion from Airway Epithelial Cells

    PubMed Central

    Adler, Kenneth B.; Tuvim, Michael J.; Dickey, Burton F.

    2013-01-01

    Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3??106?Da per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ?1??m in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the generation of DAG and of IP3 that releases calcium from apical ER. Stimulated secretion requires activation of the low affinity calcium sensor Synaptotagmin-2, while a corresponding high affinity calcium sensor in basal secretion is not known. The core exocytic machinery is comprised of the SNARE proteins VAMP8, SNAP23, and an unknown Syntaxin protein, together with the scaffolding protein Munc18b. Common and distinct features of this exocytic system in comparison to neuroendocrine cells and neurons are highlighted. PMID:24065956

  11. The effect of LRAP on enamel organ epithelial cell differentiation.

    PubMed

    Le, T Q; Zhang, Y; Li, W; Denbesten, P K

    2007-11-01

    Leucine-rich amelogenin peptide (LRAP) is an alternatively spliced amelogenin found in the developing enamel organ. LRAP functions to regulate the development of mesenchymal-derived cells; however, its effect on cells of the enamel organ remains unclear. The hypothesis tested in this study is that LRAP also regulates human enamel organ epithelial cells. Recombinant human LRAP (rH58) was synthesized in E. coli, purified, and exogenously added to cultures of human primary enamel epithelial cells, which were analyzed for changes in cell proliferation and differentiation. rH58 had no effect on cell proliferation, but altered enamel epithelial cell morphology, resulting in larger, more rounded cells. Immunofluorescence showed that rH58 treatment increased amelogenin synthesis, but down-regulated Notch1 expression in enamel epithelial cells. LAMP-1, a membrane receptor for LRAP in mesenchymal cells, was identified and was up-regulated in the presence of rH58. These results suggest that rH58 promotes differentiation of human enamel organ epithelial cells. PMID:17959903

  12. Salmonella typhi stimulation of human intestinal epithelial cells induces secretion of epithelial cell-derived interleukin-6.

    PubMed Central

    Weinstein, D L; O'Neill, B L; Metcalf, E S

    1997-01-01

    Interleukin 6 (IL-6) is a multifunctional cytokine that has been shown to be associated with both systemic and tissue-specific responses within the host. Moreover, IL-6 is produced by both lymphoid and nonlymphoid cells and has been identified as a growth-inducing, growth-inhibiting, and differentiation-inducing factor for these cells. Recent studies of uropathogenic and upper respiratory pathogens have suggested that epithelial cell-derived IL-6 plays a role in mucosal host-parasite interactions. Since many mucosal enteric pathogens enter the host through the epithelial cells of the distal small intestine, a role for intestinal epithelial cell-derived IL-6 in the initial interaction between bacteria and host might also be predicted. However, no studies to date have determined whether the interaction of any bacteria with the epithelial cells that line the small intestine of the host can induce IL-6. To address this issue, we have established an in vitro model to evaluate the capacity of the gram-negative bacterium Salmonella typhi to induce IL-6 in the small intestine epithelial cell line Int407 and in other intestinal epithelial cell lines. The results demonstrate that both wild-type and live, attenuated S. typhi vaccine strains induce small and large intestine epithelial cells to secrete IL-6, and kinetic analysis suggests that IL-6 may be one of the earliest responses following adherence and invasion of enteric organisms. Thus, these studies suggest a physiologic role for epithelial cell-derived IL-6 in the initial interactions between host and bacterium in the small intestine. PMID:9009288

  13. Fully interlocking: a story of teamwork among breast epithelial cells.

    PubMed

    Alexander, Caroline M; Joshi, Purna A; Khokha, Rama

    2014-01-27

    In this issue of Developmental Cell, Forster etal. (2014) show that the basal myoepithelial cell layer directs the final maturation of the adjacent luminal cell sheet during pregnancy. Do all mammary epithelial cells both give and take instructions from others to create the milk production machinery? PMID:24480641

  14. ONCOGENE ALTERNATIONS IN IN VITRO TRANSFORMED RAT TRACHEAL EPITHELIAL CELLS

    EPA Science Inventory

    Ten derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 non-tumorigenic cell line transformed by treatment with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP) and/or 12-0-tetradecanoylphor...

  15. 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.

  16. Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

    PubMed Central

    Maggio, Savannah; Takeda, Kazuyo; Stark, Felicity; Meierovics, Anda I.; Yabe, Idalia; Cowley, Siobhan C.

    2015-01-01

    The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates. PMID:26379269

  17. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-12-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction.

  18. 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.

  19. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    SciTech Connect

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with {sup 3}H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-{beta} did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 {times} 10{sup 6} sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined.

  20. CXCL9 Regulates TGF-?1 induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells

    PubMed Central

    OBeirne, Sarah L; Walsh, Sinead M; Fabre, Aurlie; Reviriego, Carlota; Worrell, Julie C; Counihan, Ian P; Lumsden, Robert V; Cramton-Barnes, Jennifer; Belperio, John A.; Donnelly, Seamas C; Boylan, Denise; Marchal-Somme, Jolle; Kane, Rosemary; Keane, Michael P

    2016-01-01

    Epithelial to mesenchymal transition (EMT), whereby fully differentiated epithelial cells transition to a mesenchymal phenotype has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). CXCR3 and its ligands are recognized to play a protective role in pulmonary fibrosis. In this study, we investigated the presence and extent of EMT and CXCR3 expression in human IPF surgical lung biopsies and assessed whether CXCR3 and its ligand CXCL9 modulate EMT in alveolar epithelial cells. Coexpression of the epithelial maker thyroid transcription factor-1, and mesenchymal marker ?-smooth muscle actin and CXCR3 expression was examined by immunohistochemical staining of IPF surgical lung biopsies. Epithelial and mesenchymal marker expression was examined by quantitative real-time PCR, Western blotting, and immunofluorescence in human alveolar epithelial (A549) cells treated with TGF-?1 and CXCL9, whilst Smad2, Smad3, and Smad7 expression and cellular localization examined by Western blotting. We found that significantly more cells were undergoing EMT in fibrotic versus normal areas of lung in IPF surgical lung biopsy samples. CXCR3 was expressed by type II pneumocytes and fibroblasts in fibrotic areas in close proximity to cells undergoing EMT. In vitro, CXCL9 abrogated TGF-?1 induced EMT. A decrease in TGF-?1 induced phosphorylation of Smad2 and Smad3 occurred with CXCL9 treatment. This was associated with increased shuttling of Smad7 from the nucleus to the cytoplasm where it inhibits Smad phosphorylation. This suggests a role for EMT in the pathogenesis of IPF and provides a novel mechanism for the inhibitory effects of CXCL9 on TGF-?1 induced EMT. PMID:26268659

  1. Rat glomerular epithelial cells in culture. Parietal or visceral epithelial origin

    SciTech Connect

    Norgaard, J.O.

    1987-09-01

    Isolated glomeruli from rats were explanted under standard culture conditions and outgrowths were studied by light and electron microscopy in order to identify the cells. Rat glomerular samples contained 20 to 30% structurally well-preserved encapsulated glomeruli which had a large rate of attachment to the substrate and very constantly gave rise to cellular outgrowth. In order to label cells from which outgrowth originated the glomerular incorporation of (/sup 3/H)thymidine was studied in the preattachment phase. By light and electron microscope autoradiograph it was demonstrated that label was located only over visceral and parietal epithelial cells during the first 3 days of culture. Incorporation of (/sup 3/H)thymidine was seen in mesangial cells after 5 days, i.e., after the glomeruli had attached to the culture vessels and the initial outgrowth had appeared. Consequently the first cells to grow out were of epithelial origin. Glomeruli were then incubated with (/sup 3/H)thymidine for the first 2 1/2 days of culture in order to label the epithelial cells, then were allowed to attach to the substrate and induce cell outgrowth. By light microscope autoradiography performed with the outgrowths in situ two types of cells with labeled nuclei were seen: (a) a small, polyhedral ciliated cell which grew in colonies where the cells were joined by junctional complexes (type I), and (b) a second very large, often multinucleated cell (type II). Based on the structural resemblance with their counterparts in situ and on comparisons with positively identified visceral epithelial cells in outgrowths from other species it is suggested that type I cells are derived from the parietal epithelium of Bowman's capsule and type II cells from the visceral epithelium.

  2. 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. PMID:20113446

  3. The Epithelial Cell in Lung Health and Emphysema Pathogenesis

    PubMed Central

    Mercer, Becky A.; Lemaître, Vincent; Powell, Charles A.; D’Armiento, Jeanine

    2009-01-01

    Cigarette smoking is the primary cause of the irreversible lung disease emphysema. Historically, inflammatory cells such as macrophages and neutrophils have been studied for their role in emphysema pathology. However, recent studies indicate that the lung epithelium is an active participant in emphysema pathogenesis and plays a critical role in the lung’s response to cigarette smoke. Tobacco smoke increases protease production and alters cytokine expression in isolated epithelial cells, suggesting that these cells respond potently even in the absence of a complete inflammatory program. Tobacco smoke also acts as an immunosuppressant, reducing the defense function of airway epithelial cells and enhancing colonization of the lower airways. Thus, the paradigm that emphysema is strictly an inflammatory-cell based disease is shifting to consider the involvement of resident epithelial cells. Here we review the role of epithelial cells in lung development and emphysema. To better understand tobacco-epithelial interactions we performed microarray analyses of RNA from human airway epithelial cells exposed to smoke extract for 24 hours. These studies identified differential regulation of 425 genes involved in diverse biological processes, such as apoptosis, immune function, cell cycle, signal transduction, proliferation, and antioxidants. Some of these genes, including VEGF, glutathione peroxidase, IL-13 receptor, and cytochrome P450, have been previously reported to be altered in the lungs of smokers. Others, such as pirin, cathepsin L, STAT1, and BMP2, are shown here for the first time to have a potential role in smoke-associated injury. These data broaden our understanding of the importance of epithelial cells in lung health and cigarette smoke-induced emphysema. PMID:19662102

  4. DA-6034 Induces [Ca2+]i Increase in Epithelial Cells

    PubMed Central

    Yang, Yu-Mi; Park, Soonhong; Ji, HyeWon; Kim, Tae-im; Kim, Eung Kweon; Kang, Kyung Koo

    2014-01-01

    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 Ca2+ 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 Ca2+ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca2+-activated Cl- channels (CaCCs) and increased intracellular calcium concentrations ([Ca2+]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca2+]i in mouse salivary gland cells and human corneal epithelial cells. [Ca2+]i increase of DA-6034 was dependent on the Ca2+ entry from extracellular and Ca2+ release from internal Ca2+ 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 Ca2+ stores. These results suggest that DA-6034 induces Ca2+ signaling via extracellular Ca2+ entry and RyRs-sensitive Ca2+ release from internal Ca2+ stores in epithelial cells. PMID:24757369

  5. Attachment of epithelial cells and fibroblasts to ceramic materials.

    PubMed

    Niederauer, G G; McGee, T D; Keller, J C; Zaharias, R S

    1994-04-01

    This study examined in vitro gingival epithelial and fibroblast cell attachment to ceramic materials made of tricalcium phosphate and/or magnesium aluminate spinel. The composite made of tricalcium phosphate and spinel is called 'osteoceramic'. These ceramics had various compositions and surface structures, which were initially characterized. Cell attachment assays were performed using both cell types to compare cellular response to the ceramic materials. Specimens were also prepared for scanning electron microscopy to investigate cellular morphology. The highest levels of cell attachment for gingival epithelial cells were observed on the rough osteoceramic surface, whereas gingival fibroblasts attached least to the rough osteoceramic surface. PMID:8061125

  6. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON ; 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.

  7. Epithelial cell guidance by self-generated EGF gradients

    PubMed Central

    Scherber, Cally; Aranyosi, Alexander J.; Kulemann, Birte; Thayer, Sarah P.; Toner, Mehmet; Iliopoulos, Othon

    2012-01-01

    Cancer epithelial cells often migrate away from the primary tumor to invade into the surrounding tissues. Their migration is commonly assumed to be directed by pre-existent spatial gradients of chemokines and growth factors in the target tissues. Unexpectedly however, we found that the guided migration of epithelial cells is possible in vitro in the absence of pre-existent chemical gradients. We observed that both normal and cancer epithelial cells can migrate persistently and reach the exit along the shortest path from microscopic mazes filled with uniform concentrations of media. Using microscale engineering techniques and biophysical models, we uncovered a self-guidance strategy during which epithelial cells generate their own guiding cues under conditions of biochemical confinement. The self-guidance strategy depends on the balance between three interdependent processes: epidermal growth factor (EGF) uptake by the cells (U), the restricted transport of EGF through the structured microenvironment (T), and cell chemotaxis toward the resultant EGF gradients (C). The UTC self-guidance strategy can be perturbed by inhibition of signalling through EGF-receptors and appears to be independent from chemokine signalling. Better understanding of the UTC self-guidance strategy could eventually help devise new ways for modulating epithelial cell migration and delaying cancer cell invasion or accelerating wound healing. PMID:22314635

  8. 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

  9. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

    PubMed

    Bergstralh, Dan T; Lovegrove, Holly E; St Johnston, Daniel

    2015-11-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  10. 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.

  11. Estrogen and progesterone together expand murine endometrial epithelial progenitor cells

    PubMed Central

    Janzen, DM; Cheng, D; Schafenacker, AM; Paik, DY; Goldstein, AS; Witte, ON; Jaroszewicz, A; Pellegrini, M; Memarzadeh, S

    2013-01-01

    Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM+CD44+ITGA6hiThy1−PECAM1−PTPRC−Ter119−, comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multi-lineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Co-administration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium. PMID:23341289

  12. 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.

  13. 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

  14. Cell division and the maintenance of epithelial order

    PubMed Central

    Ragkousi, Katerina

    2014-01-01

    Epithelia are polarized layers of adherent cells that are the building blocks for organ and appendage structures throughout animals. To preserve tissue architecture and barrier function during both homeostasis and rapid growth, individual epithelial cells divide in a highly constrained manner. Building on decades of research focused on single cells, recent work is probing the mechanisms by which the dynamic process of mitosis is reconciled with the global maintenance of epithelial order during development. These studies reveal how symmetrically dividing cells both exploit and conform to tissue organization to orient their mitotic spindles during division and establish new adhesive junctions during cytokinesis. PMID:25349258

  15. FOXM1 confers to epithelial-mesenchymal transition, stemness and chemoresistance in epithelial ovarian carcinoma cells

    PubMed Central

    Chiu, Wen-Tai; Huang, Yu-Fang; Tsai, Huei-Yu; Chen, Chien-Chin; Chang, Chang-Hwa; Huang, Soon-Cen; Hsu, Keng-Fu; Chou, Cheng-Yang

    2015-01-01

    Chemoresistance to anti-cancer drugs substantially reduces survival in epithelial ovarian cancer. In this study, we showed that chemoresistance to cisplatin and paclitaxel induced the epithelial-mesenchymal transition (EMT) and a stem cell phenotype in ovarian cancer cells. Chemoresistance was associated with the downregulation of epithelial markers and the upregulation of mesenchymal markers, EMT-related transcription factors, and cancer stem cell markers, which enhanced invasion and sphere formation ability. Overexpression of FOXM1 increased cisplatin-resistance and sphere formation in cisplatin-sensitive and low FOXM1-expressing ovarian cancer cells. Conversely, depletion of FOXM1 via RNA interference reduced cisplatin resistance and sphere formation in cisplatin-resistant and high FOXM1-expressing cells. Overexpression of FOXM1 also increased the expression, nuclear accumulation, and activity of β-CATENIN in chemoresistant cells, whereas downregulation of FOXM1 suppressed these events. The combination of cisplatin and the FOXM1 inhibitor thiostrepton inhibited the expression of stem cell markers in chemoresistant cells and subcutaneous ovarian tumor growth in mouse xenografts. In an analysis of 106 ovarian cancer patients, high FOXM1 levels in tumors were associated with cancer progression and short progression-free intervals. Collectively, our findings highlight the importance of FOXM1 in chemoresistance and suggest that FOXM1 inhibitors may be useful for treatment of ovarian cancer. PMID:25537512

  16. Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures.

    PubMed

    Huang, Lijia; van Loveren, Cor; Ling, Junqi; Wei, Xi; Crielaard, Wim; Deng, Dong Mei

    2016-04-01

    Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions. PMID:26963862

  17. 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.

  18. Lymphotoxin beta receptor signaling limits mucosal damage through driving IL-23 production by epithelial cells

    PubMed Central

    Macho-Fernandez, Elise; Koroleva, Ekaterina P.; Spencer, Cody M.; Tighe, Michael; Torrado, Egidio; Cooper, Andrea M.; Fu, Yang-Xin; Tumanov, Alexei V.

    2015-01-01

    The immune mechanisms regulating epithelial cell repair after injury remain poorly defined. We demonstrate here that lymphotoxin beta receptor (LTβR) signaling in intestinal epithelial cells promotes self-repair after mucosal damage. Using a conditional gene-targeted approach, we demonstrate that LTβR signaling in intestinal epithelial cells is essential for epithelial IL-23 production and protection against epithelial injury. We further show that epithelial-derived IL-23 promotes mucosal wound healing by inducing the IL-22-mediated proliferation and survival of epithelial cells and mucus production. Additionally, we identified CD4−CCR6+T-bet−RORγt+ lymphoid tissue inducer cells as the main producers of protective IL-22 after epithelial damage. Thus, our results reveal a novel role for LTβR signaling in epithelial cells in the regulation of intestinal epithelial cell homeostasis to limit mucosal damage. PMID:25183367

  19. Lymphotoxin beta receptor signaling limits mucosal damage through driving IL-23 production by epithelial cells.

    PubMed

    Macho-Fernandez, E; Koroleva, E P; Spencer, C M; Tighe, M; Torrado, E; Cooper, A M; Fu, Y-X; Tumanov, A V

    2015-03-01

    The immune mechanisms regulating epithelial cell repair after injury remain poorly defined. We demonstrate here that lymphotoxin beta receptor (LT?R) signaling in intestinal epithelial cells promotes self-repair after mucosal damage. Using a conditional gene-targeted approach, we demonstrate that LT?R signaling in intestinal epithelial cells is essential for epithelial interleukin-23 (IL-23) production and protection against epithelial injury. We further show that epithelial-derived IL-23 promotes mucosal wound healing by inducing the IL-22-mediated proliferation and survival of epithelial cells and mucus production. Additionally, we identified CD4(-)CCR6(+)T-bet(-) RAR-related orphan receptor gamma t (ROR?t)(+) lymphoid tissue inducer cells as the main producers of protective IL-22 after epithelial damage. Thus, our results reveal a novel role for LT?R signaling in epithelial cells in the regulation of intestinal epithelial cell homeostasis to limit mucosal damage. PMID:25183367

  20. Serratia marcescens internalization and replication in human bladder epithelial cells

    PubMed Central

    Hertle, Ralf; Schwarz, Heinz

    2004-01-01

    Background Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. Methods Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. Results We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. Conclusion The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections. PMID:15189566

  1. High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells.

    PubMed

    Chen, Yu-Ching; Statt, Sarah; Wu, Reen; Chang, Hao-Teng; Liao, Jiunn-Wang; Wang, Chien-Neng; Shyu, Woei-Cherng; Lee, Chen-Chen

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3? inactivation, cytoplasmic accumulation, and nuclear translocation of ?-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and ?-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3?/?-catenin signaling pathway. PMID:26739898

  2. High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells

    PubMed Central

    Chen, Yu-Ching; Statt, Sarah; Wu, Reen; Chang, Hao-Teng; Liao, Jiunn-Wang; Wang, Chien-Neng; Shyu, Woei-Cherng; Lee, Chen-Chen

    2016-01-01

    Epithelialmesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3? inactivation, cytoplasmic accumulation, and nuclear translocation of ?-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and ?-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3?/?-catenin signaling pathway. PMID:26739898

  3. Characterization of discrete equine intestinal epithelial cell lineages

    PubMed Central

    Gonzalez, Liara M.; Kinnin, Leslie A.; Blikslager, Anthony T.

    2015-01-01

    OBJECTIVE To characterize epithelial cells of the small intestine and colon in horses without clinical gastrointestinal abnormalities with an emphasis on the stem cell niche constituents. SAMPLE Mucosal biopsy specimens from small and large intestines obtained from 12 horses euthanized for reasons unrelated to gastrointestinal disease or systemic disease. PROCEDURES Intestinal biopsy specimens were collected by sharp dissection immediately following euthanasia. Specimens were prepared for immunohistochemical, immunofluorescence, and transmission electron microscopic imaging to detect and characterize each epithelial cell type. Antibodies against protein biomarkers for cellular identification were selected on the basis of expression in other mammalian species. RESULTS Intestinal epithelial cell types were identified by means of immunostaining and morphological characterization with transmission electron microscopy. Some differences in biomarker expression and antibody cross-reactivity were identified in equine tissue, compared with other species. However, each known type of mucosal epithelial cell was identified in equine tissue. CONCLUSIONS AND CLINICAL RELEVANCE The methodology used can enhance detection of stem cells and progenitor cells as well as postmitotic cell lineages in equine intestinal tissues. Results may have relevance to regenerative potential of intestinal mucosa and survival in horses with colic. PMID:25815577

  4. Internalization of Aspergillus fumigatus conidia by epithelial and endothelial cells.

    PubMed Central

    Paris, S; Boisvieux-Ulrich, E; Crestani, B; Houcine, O; Taramelli, D; Lombardi, L; Latg, J P

    1997-01-01

    The internalization of conidia of the opportunistic fungus Aspergillus fumigatus by primary cell cultures of nonprofessional phagocytes was investigated. This study is the first to show that A. fumigatus conidia were able to be engulfed by tracheal epithelial, alveolar type II, and endothelial cells. PMID:9119494

  5. 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...

  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. [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. PMID:17460896

  8. Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

    PubMed Central

    West, John D; Dorà, Natalie J; Collinson, J Martin

    2015-01-01

    In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis. PMID:25815115

  9. Clindamycin Vaginal

    MedlinePLUS

    ... an infection caused by an overgrowth of harmful bacteria in the vagina). Clindamycin is in a class ... works by slowing or stopping the growth of bacteria. Vaginal clindamycin cannot be used to treat vaginal ...

  10. Impaired oxidative phosphorylation regulates necroptosis in human lung epithelial cells.

    PubMed

    Koo, Michael Jakun; Rooney, Kristen T; Choi, Mary E; Ryter, Stefan W; Choi, Augustine M K; Moon, Jong-Seok

    2015-08-28

    Cellular metabolism can impact cell life or death outcomes. While metabolic dysfunction has been linked to cell death, the mechanisms by which metabolic dysfunction regulates the cell death mode called necroptosis remain unclear. Our study demonstrates that mitochondrial oxidative phosphorylation (OXPHOS) activates programmed necrotic cell death (necroptosis) in human lung epithelial cells. Inhibition of mitochondrial respiration and ATP synthesis induced the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and necroptotic cell death. Furthermore, we demonstrate that the activation of AMP-activated protein kinase (AMPK), resulting from impaired mitochondrial OXPHOS, regulates necroptotic cell death. These results suggest that impaired mitochondrial OXPHOS contributes to necroptosis in human lung epithelial cells. PMID:26187663

  11. Vitreous stimulates proliferation of fibroblasts and retinal pigment epithelial cells.

    PubMed

    Wiedemann, P; Ryan, S J; Novak, P; Sorgente, N

    1985-11-01

    In proliferative vitreoretinopathy, retinal pigment epithelial cells and glial cells migrate into the vitreous, proliferate, and assume characteristics of myofibroblasts. The addition of vitreous to the culture media stimulates the proliferation of porcine retinal pigment epithelial cells, bovine and lapine dermal fibroblasts but not the proliferation of bovine aortic endothelial cells and smooth muscle cells. The mitogenic activity is not species-specific, since vitreous from various species stimulates the proliferation of these cells. The mitogenic activity is destroyed by heating at 100 degrees C for 10 min or by trypsin treatment. Since the vitreous, under our assay conditions, was not mitogenic for endothelial cells or smooth muscle cells, the mitogenic activity is probably not derived from leakage into the vitreous of circulating fibroblast, epidermal or platelet-derived growth factor. PMID:4092753

  12. Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

    PubMed Central

    Maruthamuthu, Venkat; Gardel, MargaretL.

    2014-01-01

    Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering. PMID:25099795

  13. Preparation of viable single cell suspensions of tracheal epithelial cells.

    PubMed Central

    Johnson, N. F.; Margiotta, E. A.; Wilson, J. S.; Sebring, R. J.; Smith, D. M.

    1987-01-01

    This paper reports a procedure used for isolating the entire epithelial lining of the rat trachea. Isolated trachea was initially filled with 0.2% hyaluronidase and incubated at 37 degrees C for 30 min. Tracheas were flushed with medium and then reinflated with 0.5 microgram/ml cytochalasin B and re-incubated for 60 min. The tracheal lumens were again flushed and reinstilled with 24 iu/ml pronase and incubated for a further 30 min. The tracheas were flushed again and the cells removed enumerated and viability assessed by trypan blue dye exclusion. Cell yields (X 10(6)) from 30 consecutive Fischer 344 rats were 5.06 +/- 0.16 (s.e.m.) and the mean percentage of viable cells was 83.13 +/- 1.10 (s.e.m.). This cell yield was close to the estimated tracheal cell population (5.3 X 10(6)). The suspensions were predominantly single cells which apparently retained a normal ultrastructural appearance. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:3555592

  14. [The role of the epithelial cell in asthma].

    PubMed

    Mota Pinto, Anabela; Todo-Bom, Ana

    2009-01-01

    It is done a review of the intervention of the epithelial bronchial cell in the pathophysiology of asthma. The respiratory epithelium acts as a physical barrier that separates the external environment from the pulmonary internal environment. It controls the intercellular and trans -cellular permeability and this way the accessibility of the inhaled pathogens to the antigen presenting cells involved in the immuno -inflammatory response. Epithelial cells connected by tight junctions contribute to the barrier function of the airways. They express a poliovirus receiver - related protein (PRR), toll like receptors (TLRs) and protease- -activated receptors (PARs), which recognize bacterial agents and allergens. Its dysfunction turns them into important sources of inflammatory mediators. The bidirectional interaction between the epithelium and other bronchial wall elements with inhaled particles originates a structure with its own identity, the designated EMTU - Epithelial Mesenchymal Trophic Unit. These observations support a central role for the epithelial cell in chronic inflammation and in the remodelling of the asthmatic process. Infectious diseases and environmental stress can activate different cell receptors and signalling pathways that induce changes in the cell surface modifying their response to future stimulations, namely to other infectious aggressions. The bronchial epithelium has barrier functions with selective permeability; it has metabolic activity producing cytokines and chemokines stimulating the cell's recruitment and activation, increasing the bronchial reactivity and the remodelling of the airways. PMID:19401795

  15. Isolation and culture of bovine mammary epithelial stem cells.

    PubMed

    Li, Ji-Xia; Zhang, Yong; Ma, Li-Bing; Sun, Jian-Hong; Yin, Bao-Ying

    2009-01-01

    Bovine mammary epithelial stem cells (MESCs) are very important in agricultural production and bioengineering. In the present study, we compared different isolation and culture methods for MESCs and observed their growth and differentiation characteristics. MESCs have an extremely weak proliferation capacity, and it is very difficult to obtain and prolong subculture of a bovine mammary epithelial stem cell line. We obtained some multipotent MESC aggregates that looked like spherical colonies. These colonies were only derived from suspension culture and were induced to differentiate into epithelial-like cells, myoepithelial-like cells and secretory cells and to establish a ductal-like structure. In contrast, MESCs cultured in adherent culture displayed low morphogenetic competence and only differentiated into epithelial-like cells. MESCs are often identified by testing their differentiation in vivo; however, herein, we have demonstrated the in vitro differentiation potential of bovine MESCs. In our study, beta 1-integrin and alpha 6-integrin which are expressed by human epidermal stem cells, were found in bovine, which shows that bovine MESCs share the same molecular signature as human MESCs. PMID:19194071

  16. 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 m) than unmodified gels (pore diameter: 0.05-0.1 m) 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. PMID:24142706

  17. Intestinal epithelial cells and their role in innate mucosal immunity

    PubMed Central

    Maldonado-Contreras, A. L.

    2014-01-01

    The mucosal surfaces of the respiratory, gastrointestinal and urogenital tracts are covered by a layer of epithelial cells that are responsible for sensing and promoting a host immune response in order to establish the limits not only for commensal microorganisms but also for foreign organisms or particles. This is a remarkable task as the human body represents a composite of about 10 trillion human-self cells plus non-self cells from autochthonous or indigenous microbes that outnumber human cells 10:1. Hence, the homeostasis of epithelial cells that line mucosal surfaces relies on a fine-tuned immune system that patrols the boundaries between human and microbial cells. In the case of the intestine, the epithelial layer is composed of at least six epithelial cell lineages that act as a physiological barrier in addition to aiding digestion and the absorption of nutrients, water and electrolytes. In this review, we highlight the immense role of the intestinal epithelium in coordinating the mucosal innate immune response. PMID:21104188

  18. Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection

    PubMed Central

    Ju, Chia-Hsin; Chockalingam, Annapoorani; Leifer, Cynthia A.

    2012-01-01

    The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii (T. gondii) is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from intraperitoneal infection models, more recent studies have revealed the importance of studying immunity following infection through the natural per-oral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-κB nuclear translocation, and secretion of interleukin (IL)-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses. PMID:19917706

  19. Viscoelasticity of human alveolar epithelial cells subjected to stretch.

    PubMed

    Trepat, Xavier; Grabulosa, Mireia; Puig, Ferranda; Maksym, Geoffrey N; Navajas, Daniel; Farr, Ramon

    2004-11-01

    Alveolar epithelial cells undergo stretching during breathing and mechanical ventilation. Stretch can modify cell viscoelastic properties, which may compromise the balance of forces in the alveolar epithelium. We studied the viscoelasticity of alveolar epithelial cells (A549) subjected to equibiaxial distention with a novel experimental approach. Cells were cultured on flexible substrates and subjected to stepwise deformations of up to 17% with a device built on an inverted microscope. Simultaneously, cell storage (G') and loss (G'') moduli were measured (0.1-100 Hz) with optical magnetic twisting cytometry. G' and G'' increased with strain up to 64 and 30%, respectively, resulting in a decrease in G''/G' (15%). This stretch-induced response was inhibited by disruption of the actin cytoskeleton with latrunculin A. G' increased with frequency following a power law with exponent alpha = 0.197. G'' increased proportionally to G' but exhibited a more marked frequency dependence at high frequencies. Stretching (14%) caused a fall in alpha (13%). At high stretching amplitudes, actual cell strain (14.4%) was lower than the applied substrate strain (17.3%), which could indicate a partial cell detachment. These data suggest that cytoskeletal prestress modulates the elastic and frictional properties of alveolar epithelial cells in a coupled manner, according to soft glassy rheology. Stretch-induced cell stiffening could compromise the balance of forces at the cell-cell and cell-matrix adhesions. PMID:15246973

  20. Apical trafficking in epithelial cells: signals, clusters and motors

    PubMed Central

    Weisz, Ora A.; Rodriguez-Boulan, Enrique

    2009-01-01

    Summary In the early days of epithelial cell biology, researchers working with kidney and/or intestinal epithelial cell lines and with hepatocytes described the biosynthetic and recycling routes followed by apical and basolateral plasma membrane (PM) proteins. They identified the trans-Golgi network and recycling endosomes as the compartments that carried out apical-basolateral sorting. They described complex apical sorting signals that promoted association with lipid rafts, and simpler basolateral sorting signals resembling clathrin-coated-pit endocytic motifs. They also noticed that different epithelial cell types routed their apical PM proteins very differently, using either a vectorial (direct) route or a transcytotic (indirect) route. Although these original observations have generally held up, recent studies have revealed interesting complexities in the routes taken by apically destined proteins and have extended our understanding of the machinery required to sustain these elaborate sorting pathways. Here, we critically review the current status of apical trafficking mechanisms and discuss a model in which clustering is required to recruit apical trafficking machineries. Uncovering the mechanisms responsible for polarized trafficking and their epithelial-specific variations will help understand how epithelial functional diversity is generated and the pathogenesis of many human diseases. PMID:19923269

  1. Keratins are novel markers of renal epithelial cell injury.

    PubMed

    Djudjaj, Sonja; Papasotiriou, Marios; Bülow, Roman D; Wagnerova, Alexandra; Lindenmeyer, Maja T; Cohen, Clemens D; Strnad, Pavel; Goumenos, Dimitrios S; Floege, Jürgen; Boor, Peter

    2016-04-01

    Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress. PMID:26924053

  2. Characterization of a Hormone-Responsive Organotypic Human Vaginal Tissue Model: Morphologic and Immunologic Effects.

    PubMed

    Ayehunie, Seyoum; Islam, Ayesha; Cannon, Chris; Landry, Timothy; Pudney, Jeffrey; Klausner, Mitchell; Anderson, Deborah J

    2015-08-01

    Estrogen and progesterone regulate proliferation and differentiation of epithelial cells in the female genital tract. We investigated the effects of these hormones on reconstructed human organotypic vaginal epithelial tissue models (EpiVaginal). We ascertained that epithelial cells in the tissue models express estrogen and progesterone receptors. Treatment with estradiol-17? (E(2)) significantly increased epithelium thickness and transepithelial electrical resistance (TEER), whereas progesterone (P) treatment resulted in thinning of the epithelium and decreased TEER when compared with untreated controls. Exposure to E(2) increased (1) the expression of the progesterone receptor B (PR-B), (2) accumulation of glycogen in suprabasal cells, (3) epithelial differentiation, and (4) the expression of a number of gene pathways associated with innate immunity, epithelial differentiation, wound healing, and antiviral responses. These findings indicate that EpiVaginal tissues are hormone responsive and can be used to study the role of female reproductive hormones in innate immune responses, microbial infection, and drug delivery in the vaginal mucosa. PMID:25676577

  3. Sodium Dodecyl Sulfate and C31G as Microbicidal Alternatives to Nonoxynol 9: Comparative Sensitivity of Primary Human Vaginal Keratinocytes

    PubMed Central

    Krebs, Fred C.; Miller, Shendra R.; Catalone, Bradley J.; Welsh, Patricia A.; Malamud, Daniel; Howett, Mary K.; Wigdahl, Brian

    2000-01-01

    A broad-spectrum vaginal microbicide must be effective against a variety of sexually transmitted disease pathogens and be minimally toxic to the cell types found within the vaginal epithelium, including vaginal keratinocytes. We assessed the sensitivity of primary human vaginal keratinocytes to potential topical vaginal microbicides nonoxynol-9 (N-9), C31G, and sodium dodecyl sulfate (SDS). Direct immunofluorescence and fluorescence-activated cell sorting analyses demonstrated that primary vaginal keratinocytes expressed epithelial cell-specific keratin proteins. Experiments that compared vaginal keratinocyte sensitivity to each agent during a continuous, 48-h exposure demonstrated that primary vaginal keratinocytes were almost five times more sensitive to N-9 than to either C31G or SDS. To evaluate the effect of multiple microbicide exposures on cell viability, primary vaginal keratinocytes were exposed to N-9, C31G, or SDS three times during a 78-h period. In these experiments, cells were considerably more sensitive to C31G than to N-9 or SDS at lower concentrations within the range tested. When agent concentrations were chosen to result in an endpoint of 25% viability after three daily exposures, each exposure decreased cell viability at the same constant rate. When time-dependent sensitivity during a continuous 48-h exposure was examined, exposure to C31G for 18 h resulted in losses in cell viability not caused by either N-9 or SDS until at least 24 to 48 h. Cumulatively, these results reveal important variations in time- and concentration-dependent sensitivity to N-9, C31G, or SDS within populations of primary human vaginal keratinocytes cultured in vitro. These investigations represent initial steps toward both in vitro modeling of the vaginal microenvironment and studies of factors that impact the in vivo efficacy of vaginal topical microbicides. PMID:10858360

  4. Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation

    PubMed Central

    Juncadella, Ignacio J.; Kadl, Alexandra; Sharma, Ashish K.; Shim, Yun M.; Hochreiter-Hufford, Amelia; Borish, Larry; Ravichandran, Kodi S.

    2013-01-01

    Lung epithelial cells can influence immune responses to airway allergens1,2. Airway epithelial cells also undergo apoptosis after encountering environmental allergens3; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells1,4,5. Administration of exogenous IL-10 ‘rescued’ the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens. PMID:23235830

  5. Intestinal epithelial cell regulation of mucosal inflammation.

    PubMed

    Yu, Yimin; Sitaraman, Shanthi; Gewirtz, Andrew T

    2004-01-01

    The intestinal epithelium serves as one of human's primary interfaces with the outside world. This interface is very heavily colonized with bacteria and yet permits absorption of life-sustaining nutrients while protecting the tissues below from microbial onslaught. Although the gut epithelium had been classically thought to achieve this function primarily by functioning as a passive, albeit highly selective, barrier, research over the last decade has demonstrated that in fact the epithelium plays a very active role in protecting the host from the bacteria that colonize it. As a consequence of its mediation of mucosal immunity, intestinal epithelial dysfunction appears to be central to diseases associated with aberrant gut mucosal immune responses such as inflammatory bowel disease (IBD). This article reviews: (1) how the gut epithelium participates in regulating innate immune inflammatory responses to enteric pathogens, (2) how these responses may regulate the adaptive immune system, (3) mechanisms that may resolve acute inflammation, and (4) how epithelial dysfunction may participate in regulating both the active and chronic phases of IBD. PMID:15181270

  6. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells

    PubMed Central

    Padilla-Nash, Hesed M.; McNeil, Nicole E.

    2013-01-01

    Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research. PMID:23619298

  7. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells.

    PubMed

    Padilla-Nash, Hesed M; McNeil, Nicole E; Yi, Ming; Nguyen, Quang-Tri; Hu, Yue; Wangsa, Danny; Mack, David L; Hummon, Amanda B; Case, Chanelle; Cardin, Eric; Stephens, Robert; Difilippantonio, Michael J; Ried, Thomas

    2013-08-01

    Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research. PMID:23619298

  8. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-11-10

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.

  9. Epithelial progenitor cells in the rat trachea

    SciTech Connect

    Johnson, N.F.; Hubbs, A.F. )

    1990-01-01

    Highly pure populations of basal and secretory cells from the rat trachea have been inoculated into denuded tracheal grafts to determine the differentiation pathways of these two cell types. The basal cell inoculum resulted in an epithelium comprised of only basal and ciliated cells, while the secretory cell inoculum gave rise to an epithelium comprised of secretory, basal, and ciliated cells. These results show that, in the rat trachea, the secretory cell is the major progenitorial cell type and the basal cell has only limited progenitorial capacity.

  10. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

    Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

  11. Lung epithelial cell death induced by oil-dispersant mixtures.

    PubMed

    Wang, He; Shi, Yongli; Major, Danielle; Yang, Zhanjun

    2012-08-01

    The dispersants used in oil spill disasters are claimed to be safe, but increased solubility of high-molecular-weight components in crude oil is of public health concern. The water-accommodated fractions (WAF) of crude oil mixed with dispersants may become airborne and cause lung epithelial damage when inhaled. This study was designed to examine the cell death and related death pathways of lung epithelial cells in response to WAF. Cultured A549 cells were treated for 2 or 24h with different concentrations of WAF. The WAF was prepared by mixing each of the dispersants (Corexit EC9527A, Corexit EC9500A and Corexit EC9580A) with crude oil for extraction with PBS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay, lactate dehydrogenase assay, morphology and cleaved caspase 9 protein, and microtubule-associated protein 1 light chain 3 were all used to measure cell viability, necrosis, apoptosis and autophagy quantitation, respectively. Results showed that the WAF of oil-dispersant mixtures caused cell death in the lung epithelial cells, in a dose-dependent manner, with the major cellular pathways of necrosis and apoptosis involved. Autophagy also occurred in cells exposed to WAF mixtures at lower concentrations before any detectable cell death, indicating greater sensitivity to WAF exposure. The three types of cell behavior, namely necrosis, apoptosis and autophagy, may play different roles in oil spill-related respiratory disorders. PMID:22504303

  12. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells.

    PubMed

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-03-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel. PMID:23538640

  13. 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?m-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?m-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. Copyright 2012 John Wiley & Sons, Ltd. PMID:23239605

  14. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  15. Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism.

    PubMed

    Mathow, Daniel; Chessa, Federica; Rabionet, Mariona; Kaden, Sylvia; Jennemann, Richard; Sandhoff, Roger; Grne, Hermann-Josef; Feuerborn, Alexander

    2015-03-01

    This study proposes that the transcription factor Zeb1 modulates epithelial cell adhesion by diverting glycosphingolipid metabolism. Zeb1 promotes expression of a-series glycosphingolipids via regulating expression of GM3 synthase (St3gal5), which mechanistically involves Zeb1 binding to the St3gal5 promoter as well as suppressing microRNA-mediated repression of St3gal5. Functionally, the repression of St3gal5 suffices to elevate intercellular adhesion and expression of distinct junction-associated proteins, reminiscent of knockdown of Zeb1. Conversely, overexpressing St3gal5 sensitizes cells towards TGF-?1-induced disruption of cell-cell interaction and partially antagonizes elevation of intercellular adhesion imposed by Zeb1 knockdown. These results highlight a direct connection of glycosphingolipid metabolism and epithelial cell adhesion via Zeb1. PMID:25643708

  16. Structural anomalies of highly malignant respiratory tract epithelial cells

    SciTech Connect

    Manger, R.L.; Heckman, C.A.

    1982-11-01

    These studies were designed to determine whether cytostructural changes were related to malignancy and the loss of growth control in epithelial cells. Three highly malignant cell lines were derived from transplantable carcinomas of the respiratory tract and compared with three respiratory tract epithelial lines of negligible malignancy. Keratin cytoskeletons were visualized by indirect immunofluorescence staining, and sample photomicrographs representing each line were prepared. The criteria used in making the classifications to identify the features common to the highly malignant lines included the nonuniform spacing of cells in the field of view, the cell shape, and the presence of nonfluorescent areas in the lamellar cytoplasm. Since the nonuniformity of keratin distribution in the periphery of the malignant cells suggested a structural anomaly, the cell lines were also examined by scanning electron microscopy. Unlike cells from the lines of negligible malignancy, cells from two of the highly malignant lines showed thickenings in the subterminal portions of the lamellar cytoplasm. The results suggested that specific architectural changes at the cellular level might be linked to the process of epithelial transformation and tumor progression.

  17. Lactobacillus equigenerosi Strain Le1 Invades Equine Epithelial Cells

    PubMed Central

    Botha, Marlie; Botes, Marelize; Loos, Ben; Smith, Carine

    2012-01-01

    Lactobacillus equigenerosi strain Le1, a natural inhabitant of the equine gastrointestinal tract, survived pH 3.0 and incubation in the presence of 1.5% (wt/vol) bile salts for at least 2 h. Strain Le1 showed 8% cell surface hydrophobicity, 60% auto-aggregation, and 47% coaggregation with Clostridium difficile C6. Only 1% of the cells adhered to viable buccal epithelial cells and invaded the cells within 20 min after contact. Preincubation of strain Le1 in a buffer containing pronase prevented adhesion to viable epithelial cells. Preincubation in a pepsin buffer delayed invasion from 20 min to 1 h. Strain Le1 did not adhere to nonviable epithelial cells. Administration of L. equigenerosi Le1 (1 × 109 CFU per 50 kg body weight) to healthy horses did not increase white blood cell numbers. Differential white blood cell counts and aspartate aminotransferase levels remained constant. Glucose, lactate, cholesterol, and urea levels remained constant during administration with L. equigenerosi Le1 but decreased during the week after administration. PMID:22504808

  18. Keratin cytoskeletons in epithelial cells of internal organs.

    PubMed

    Sun, T T; Shih, C; Green, H

    1979-06-01

    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. PMID:111242

  19. Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

    PubMed

    Zhou, Qingjun; Chen, Peng; Di, Guohu; Zhang, Yangyang; Wang, Yao; Qi, Xia; Duan, Haoyun; Xie, Lixin

    2015-05-01

    Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. PMID:25546438

  20. [Stem cell factor production from cultured nasal epithelial cells--effect on SCF production by drugs].

    PubMed

    Koyama, Mamoru; Otsuka, Hirokuni; Kusumi, Taeko; Yamauchi, Yoko

    2002-02-01

    We studied whether epithelial cells cultured in serum-free medium contained other cells or not, there were differences in SCF production from cultured nasal epithelial cells between groups of nonallergic and allergic patients, and among degrees of serum mite-CAP RAST classes of allergic patients, and how drugs inhibited SCF production. As a result, no other contaminating cells except mast cell existed in cultured cells. There was a significant difference in SCF production of cultured cells between nonallergic and class 1-2, 3-4, 5-6, and between class 1-2 and 3-4, 5-6 of mite CAP-RAST class. Cyclosporin, prednisolone, fluticasone, ketotifen, and clemastine inhibited SCF production from cultured epithelial cells, but cromoglicate and suplatast did not. Inhibition means the reduction of SCF from cells, not the growth of cultured nasal epithelial cells. PMID:11905054

  1. Thrombin promotes epithelial ovarian cancer cell invasion by inducing epithelial-mesenchymal transition

    PubMed Central

    Zhong, Yi-Cun; Zhang, Ting; Di, Wen

    2013-01-01

    Objective Over-expression of thrombin in ovarian cancer cells is associated with poor prognosis. In this study, we investigated the role of thrombin in inducing epithelial-mesenchymal transition (EMT) in SKOV3 epithelial ovarian cancer cells. Methods After thrombin treatment SKOV3 cells were subjected to western blots, reverse-transcription PCR, and enzyme-linked immunosorbent assay to quantify EMT-related proteins, mRNA expression of SMAD2, DKK1, and sFRP1, and the secretion of matrix metalloproteinases (MMPs) and cytokines. Meanwhile, invasion ability was evaluated using transwell assays. Results The results indicated a dose- and time-dependent down-regulation of E-cadherin and upregulation of N-cadherin and vimentin in thrombin-treated SKOV3 cells, compared with the thrombin-free control group (p<0.05). There was a dose- and time-dependent increase in the levels of SMAD2 and DKK1 mRNAs and a decrease in the levels of sFRP1 mRNA in thrombin-treated SKOV3 cells compared to control cells (p<0.05). Thrombin-treated SKOV3 cells exhibited increased secretion of MMP-9, MMP-2, interleukin (IL)-8, and IL-6 and increased invasion compared to untreated cells (p<0.05). Thrombin altered the morphology of SKOV3 cells to a spindle-like phenotype. Addition of hirudin to thrombin-treated cells reversed the effects of thrombin. Conclusion Thrombin induced EMT and promoted the invasion of SKOV3 cells, possibly via distinct signaling pathways. Hirudin inhibited the effects of thrombin, suggesting that anticoagulant therapy could be a novel therapeutic strategy for ovarian carcinoma. PMID:23875077

  2. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    SciTech Connect

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang Zhang, Yi

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  3. 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. PMID:26649819

  4. Modulation of epithelial tissue and cell migration by microgrooves.

    PubMed

    Dalton, B A; Walboomers, X F; Dziegielewski, M; Evans, M D; Taylor, S; Jansen, J A; Steele, J G

    2001-08-01

    We used a polystyrene substratum to study the response of migrating epithelium to 1- or 5-microm depth microgrooves with groove/ridge widths of 1, 2, 5, or 10 microm. The migration of a tissue sheet was enhanced along the microgrooves, while migration across the microgrooves was inhibited. Changing the depth of the microgrooves had a greater effect on migration than alteration of the groove/ridge width. The migration of epithelial cells from a confluent monolayer culture followed a similar pattern to that of intact epithelial tissue. Cellular extensions generally followed the microgroove direction by tracking along the top of the ridges or following the ridge walls, as revealed by scanning electron microscopy. Actin filaments within the basal cell layer of the tissue were aligned with the microgrooves, unlike filaments in the superficial layers that did not appear to be affected by the presence of underlying microgrooves. The basal cell layer of the tissue conformed to the contours of the microgroove following migration. However, the ultrastructure of the tissue above the ridges resembled that of tissue on a flat surface. We concluded that surface microgrooves have the potential to direct the migration of immediately adjacent epithelial tissue, the effect of which is to guide epithelial tissue on the surface of implanted biomaterials. PMID:11340589

  5. Adrenomedullin Expression by Gastric Epithelial Cells in Response to Infection

    PubMed Central

    Allaker, Robert P.; Kapas, Supriya

    2003-01-01

    Many surface epithelial cells express adrenomedullin, a multifunctional peptide found in a wide number of body and cell systems. Recently, we and others have proposed that adrenomedullin has an important novel role in host defense. This peptide has many properties in common with other cationic antimicrobial peptides, including the human β-defensins. Upon exposure of human gastric epithelial cells to viable cells of invasive or noninvasive strains of Helicobacter pylori, Escherichia coli, Salmonella enterica, or Streptococcus bovis, a significant increase in adrenomedullin secretion from these cells was demonstrated. Adrenomedullin gene expression was also increased in response to these microorganisms. Similar observations were noted when these cells were incubated with proinflammatory cytokines such as interleukin 1α (IL-1α), IL-6, tumor necrosis factor alpha and lipopolysaccharide. In cultured cells and an animal infection model, increased adrenomedullin peptide and gene expression was demonstrated when exposed to E. coli or Mycobacterium paratuberculosis, respectively. The data suggest there is a strong association between epithelial infection, inflammation, and adrenomedullin expression, which may have clinical relevance. The regulation of adrenomedullin expression may have therapeutic applications, such as improving or enhancing mucosal immunity. PMID:12853384

  6. Bidirectional entry of poliovirus into polarized epithelial cells.

    PubMed

    Tucker, S P; Thornton, C L; Wimmer, E; Compans, R W

    1993-01-01

    The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut. PMID:8380076

  7. Uranium induces oxidative stress in lung epithelial cells.

    PubMed

    Periyakaruppan, Adaikkappan; Kumar, Felix; Sarkar, Shubhashish; Sharma, Chidananda S; Ramesh, Govindarajan T

    2007-06-01

    Uranium compounds are widely used in the nuclear fuel cycle, antitank weapons, tank armor, and also as a pigment to color ceramics and glass. Effective management of waste uranium compounds is necessary to prevent exposure to avoid adverse health effects on the population. Health risks associated with uranium exposure includes kidney disease and respiratory disorders. In addition, several published results have shown uranium or depleted uranium causes DNA damage, mutagenicity, cancer and neurological defects. In the current study, uranium toxicity was evaluated in rat lung epithelial cells. The study shows uranium induces significant oxidative stress in rat lung epithelial cells followed by concomitant decrease in the antioxidant potential of the cells. Treatment with uranium to rat lung epithelial cells also decreased cell proliferation after 72 h in culture. The decrease in cell proliferation was attributed to loss of total glutathione and superoxide dismutase in the presence of uranium. Thus the results indicate the ineffectiveness of antioxidant system's response to the oxidative stress induced by uranium in the cells. PMID:17124605

  8. Uranium induces oxidative stress in lung epithelial cells

    PubMed Central

    Periyakaruppan, Adaikkappan; Kumar, Felix; Sarkar, Shubhashish; Sharma, Chidananda S.

    2009-01-01

    Uranium compounds are widely used in the nuclear fuel cycle, antitank weapons, tank armor, and also as a pigment to color ceramics and glass. Effective management of waste uranium compounds is necessary to prevent exposure to avoid adverse health effects on the population. Health risks associated with uranium exposure includes kidney disease and respiratory disorders. In addition, several published results have shown uranium or depleted uranium causes DNA damage, mutagenicity, cancer and neurological defects. In the current study, uranium toxicity was evaluated in rat lung epithelial cells. The study shows uranium induces significant oxidative stress in rat lung epithelial cells followed by concomitant decrease in the antioxidant potential of the cells. Treatment with uranium to rat lung epithelial cells also decreased cell proliferation after 72 h in culture. The decrease in cell proliferation was attributed to loss of total glutathione and superoxide dismutase in the presence of uranium. Thus the results indicate the ineffectiveness of antioxidant system’s response to the oxidative stress induced by uranium in the cells. PMID:17124605

  9. Iron upregulates melanogenesis in cultured retinal pigment epithelial cells

    PubMed Central

    Wolkow, Natalie; Li, Yafeng; Maminishkis, Arvydas; Song, Ying; Alekseev, Oleg; Iacovelli, Jared; Song, Delu; Lee, Jennifer C.; Dunaief, Joshua L.

    2014-01-01

    The purpose of our studies was to examine the relationship between iron and melanogenesis in retinal pigment epithelial cells, as prior observations had suggested that iron may promote melanogenesis. This relationship has potential clinical importance, as both iron overload and hyperpigmentation are associated with age-related macular degeneration (AMD). Human fetal retinal pigment epithelial cells and ARPE-19 cells were treated with iron in the form of ferric ammonium citrate, after which quantitative RT-PCR and electron microscopy were performed. Melanogenesis genes tyrosinase, tyrosinase-related protein 1, Hermansky-Pudlak Syndrome 3, premelanosome protein and dopachrome tautomerase were upregulated, as was the melanogenesis-controlling transcription factor, microphthalmia-associated transcription factor (MITF). Iron-treated cells had increased pigmentation and melanosome number. Multiple transcription factors upstream of MITF were upregulated by iron. PMID:25277027

  10. Epithelial-Mesenchymal Transition and Cell Cooperativity in Metastasis

    PubMed Central

    Tsuji, Takanori; Ibaragi, Soichiro; Hu, Guo-fu

    2009-01-01

    The role of epithelial-mesenchymal transition (EMT) in metastasis remains to be controversial. EMT has been postulated as an absolute requirement for tumor invasion and metastasis. Three different models including incomplete EMT, mesenchymal-epithelial transition (MET), and collective migration have been proposed for the role of EMT in cancer invasion and metastasis. However, skepticism remains as to whether EMT truly occurs during caner progression, and if it does, whether it plays an indispensible role in metastasis. Our recent findings suggest that EMT cells are responsible for degrading the surrounding matrix to enable invasion and intravasation of both EMT and non-EMT cells. Only non-EMT cell that have entered the blood stream are able to reestablish colonies in the secondary sites. Here we discuss an alternative model for the role of EMT in cancer metastasis in which EMT and non-EMT cells cooperate to complete the entire process of spontaneous metastasis. PMID:19738043

  11. Salmonella enterica: living a double life in epithelial cells.

    PubMed

    Knodler, Leigh A

    2015-02-01

    Intracellular bacterial pathogens can occupy a membrane-bound vacuole or live freely within the cytosol of mammalian cells. Many studies have shown that the enteric bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium), is a vacuolar pathogen. Recent data, however, have revealed that within epithelial cells there are subpopulations of vacuolar and cytosolic Salmonella. Release from the Salmonella-containing vacuole leads to transcriptional reprogramming of bacteria and their robust replication in the cytosol. Eventually, epithelial cell death via pyroptosis results in cell lysis, proinflammatory cytokine release and escape of the cytosolic bacteria into the extracellular space, providing a potential mechanism of dissemination. This review focuses on the current understanding of this newly described intracellular population of Salmonella. PMID:25461569

  12. Melanosome Motility in Fish Retinal Pigment Epithelial (RPE) Cells.

    PubMed

    King-Smith, Christina

    2016-01-01

    Several model systems have been developed to investigate mechanism and regulation of intracellular organelle motility. The fish retinal pigment epithelial (RPE) cell represents a novel yet simple system for the study of organelle motility. Primary cultures of dissociated RPE cells are easily prepared and amenable to motility studies. In vivo, melanin-containing pigment granules (melanosomes) within fish RPE migrate distances up to 100 ?m in response to light flux. When dissociated from the epithelial layer and cultured in vitro, RPE cells attach to the substrate with the apical projections extending radially from the central cell body. Melanosomes can be chemically triggered to aggregate or disperse throughout the projections, and are easily observed using phase contrast microscopy. Melanosome migration in RPE apical projections is dependent on actin filaments, and thus renders this model system useful for investigations of actin-dependent organelle motility. PMID:26498793

  13. COMPARISON OF PM-INDUCED GENE EXPRESSION PROFILES BETWEEN BRONCHIAL EPITHELIAL CELLS AND NASAL EPITHELIAL CELLS IN HUMAN

    EPA Science Inventory

    Epidemiologic studies have linked exposures to particulate matter (PM) and increased pulmonary mortality and morbidity. Bronchial epithelial cells (BEC) are the primary target of PM. PM exposure induces a wide array of biological responses in BEC. Primary human BEC, however, need...

  14. Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.

    PubMed

    Katikireddy, Kishore Reddy; Jurkunas, Ula V

    2016-01-01

    From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells. PMID:25762299

  15. Review: Corneal epithelial stem cells, their niche and wound healing

    PubMed Central

    2013-01-01

    Stem cells emerged as a concept during the second half of 19th century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemets membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  16. Epithelial stem cells in adult skin.

    PubMed

    Tadeu, Ana Mafalda Baptista; Horsley, Valerie

    2014-01-01

    The skin is the first line of defense against dehydration and external environmental aggressions. It constantly renews itself throughout adult life mainly due to the activity of tissue-specific stem cells. In this review, we discuss fundamental characteristics of different stem cell populations within the skin and how they are able to contribute to normal skin homeostasis. We also examine the most recent results regarding the cell-intrinsic and -extrinsic components of the stem cell niche within the adult skin epithelium. Finally, we address the recent efforts to understand how abnormal regulation of stem cell activity contributes to the initiation and progression of skin-associated cancers. PMID:24439804

  17. Renal epithelial cells produce and spread HIV-1 via T-Cell Contact

    PubMed Central

    BLASI, Maria; BALAKUMARAN, Bala; CHEN, Ping; NEGRI, Donatella RM; CARA, Andrea; CHEN, Benjamin K.; KLOTMAN, Mary E.

    2015-01-01

    Objective Increasing evidence supports the role of the kidney as a viral reservoir for HIV-1. In vitro co-cultivation of HIV-infected T cells with renal epithelial cells results in virus transfer to the latter, while cell free virus infection of these cells is very inefficient. In this study we further characterized the fate of HIV-1 after it is internalized in renal epithelial cells. Methods Primary or immortalized CD4+ cells were infected with a GFP-expressing replication competent HIV-1. HIV-1 transfer from T cells to epithelial cells was carried out in a co-culture system and evaluated by FACS analysis. HIV-1 integration in renal epithelial cells was evaluated by Alu-PCR and the production of infectious particles was assessed by p24-ELISA and TZM-bl assay. HIV-infected renal cells were used as donor cells in a co-culture system to evaluate their ability to transfer the virus back to T cells. Results Renal epithelial cells become productively infected by HIV-1 and multiple copies of HIV-1 can be transferred from infected T cells to renal epithelial cells. Two separate cells populations were identified among infected renal cells based on the reporter gene GFP expression level (low vs high), with only the high showing sensitivity to AZT and Ritonavir. Co-cultivation of HIV-1 infected renal cells with non-infected T cells resulted in HIV-1 transmission to T cells, supporting bidirectional exchange of virus between T cells and kidney-derived cells. Conclusions These results support the kidney as a potential reservoir where virus is exchanged between interstitial T cells and renal tubule epithelial cells. PMID:25062092

  18. Vangl2 Regulates E-Cadherin in Epithelial Cells

    PubMed Central

    Nagaoka, Tadahiro; Inutsuka, Ayumu; Begum, Khadiza; hafiz, Khandakar musabbir bin; Kishi, Masashi

    2014-01-01

    E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2Lpt/+ mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions. PMID:25373475

  19. Vangl2 regulates E-cadherin in epithelial cells.

    PubMed

    Nagaoka, Tadahiro; Inutsuka, Ayumu; Begum, Khadiza; Bin hafiz, Khandakar musabbir; Kishi, Masashi

    2014-01-01

    E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2(Lpt/+) mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions. PMID:25373475

  20. A novel method for preserving cultured limbal epithelial cells

    PubMed Central

    Utheim, Tor Paaske; Raeder, Sten; Utheim, ygunn Aass; Cai, Yiqing; Roald, Borghild; Drolsum, Liv; Lyberg, Torstein; Nicolaissen, Bjrn

    2007-01-01

    Aim To investigate organ culture preservation of cultured limbal epithelial cells in order to enhance the availability of tissue?engineered epithelia that are used to treat patients with limbal stem cell deficiency. Methods Limbal epithelial cells were cultured for 3?weeks on intact amniotic membrane fastened to a polyester membrane carrier. The cultured epithelia were stored for 1?week at 23C in organ culture medium. The preserved epithelia were then examined using a colorimetric cell viability assay, light microscopy and immunohistochemistry. Results The viability of the preserved epithelia was 84% (20%), and no statistically significant difference was found compared with non?preserved epithelia. In general, the cell borders were maintained, the nuclei showed no sign of degeneration, and the original layered structure was preserved. Mild intercellular oedema was occasionally observed. Expression of p63, K19 and vimentin was maintained. Conclusions Cultured limbal epithelial cells can be preserved in organ culture medium for 1?week at room temperature, while maintaining the original layered structure and undifferentiated phenotype. PMID:17124242

  1. Functional interleukin-2 receptors on intestinal epithelial cells.

    PubMed Central

    Ciacci, C; Mahida, Y R; Dignass, A; Koizumi, M; Podolsky, D K

    1993-01-01

    The presence of receptors for the cytokine IL-2 was assessed in the IEC-6 cell line established from normal rat crypt epithelium and primary intestinal epithelial cells. 125I-IL-2 was found to specifically bind to subconfluent IEC-6 cells. Maximal binding was observed within 30 min after addition of the ligand; binding could be inhibited by excess unlabeled IL-2 or addition of antibody to the IL-2 receptor. Both intermediate and low affinity receptors with approximate Kd of 10 and 100 pM, respectively were present. Kinetic analysis were consistent with the results of Western blot analysis using an antisera to the 75-kD IL-2 receptor beta chain. IL-2 receptors appeared to be functional; addition of IL-2 led to modulation of proliferation with initial stimulation at 24 h followed by inhibition at 48 h. This effect could be blocked by addition of antibody to the IL-2 receptor beta chain. IL-2 treatment could be shown to enhance expression (range = 4- to 50-fold stimulation) of TGF-beta, as well as the lectin protein mac-2, in IEC-6 cells. The relevance of observations in the IEC-6 cell line to intestinal mucosa in vivo was supported by the demonstration of a gradient of expression of the IL-2 receptor in primary rat intestinal epithelial cells by Western blot analysis. In addition, mRNA for the IL-2 receptor-beta chain was demonstrated by Northern blot analysis using mRNA from primary rat intestinal epithelial cells depleted of detectable contaminating intraepithelial lymphocytes by two cycles of fractionation on Percoll gradients. Collectively, these observations suggest that the range of cellular targets of the putative lymphokine IL-2 is broader than appreciated, and IL-2 may serve to integrate epithelial and lymphocyte responses in the intestinal mucosa. Images PMID:8326018

  2. Cytokeratin changes in cell culture systems of epithelial cells isolated from oral mucosa: a short review.

    PubMed

    Gasparoni, Alberto; Squier, Christopher Alan; Fonzi, Luciano

    2005-01-01

    In the past three decades, many studies have analyzed ultrastructural and molecular markers of differentiation in squamous stratified epithelial tissues. In these tissues, epithelial cells migrating from the basal layer to the upper layers undergo drastic changes, which involve membrane-associated proteins, DNA synthesis, phenotypic aspects, lipid composition, and cytoskeletal components. Cytoskeletal components include a large and heterogeneous group, including intermediate filaments, components of the cornified envelope, and of the stratum corneum. When grown in mono- and multilayer cell cultures, epithelial cells isolated from the oral mucosa may reproduce many of the biochemical and morphological aspects of epithelial tissue in vivo. In the present paper, we examine phenotypic changes, development of suprabasal layer, and Involucrin expression occurring in differentiating oral epithelial cells, based on literature review and original data. PMID:16277157

  3. Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics

    PubMed Central

    Blume, Cornelia; Reale, Riccardo; Held, Marie; Millar, Timothy M.; Collins, Jane E.; Davies, Donna E.; Morgan, Hywel; Swindle, Emily J.

    2015-01-01

    The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL–8 release is detectable within the first 2h and peaks at 4–6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms. PMID:26436734

  4. The Role of Vaginal Brachytherapy in the Treatment of Surgical Stage I Papillary Serous or Clear Cell Endometrial Cancer

    SciTech Connect

    Barney, Brandon M.; Petersen, Ivy A.; Mariani, Andrea; Dowdy, Sean C.; Bakkum-Gamez, Jamie N.; Haddock, Michael G.

    2013-01-01

    Objectives: The optimal adjuvant therapy for International Federation of Gynecology and Obstetrics (FIGO) stage I papillary serous (UPSC) or clear cell (CC) endometrial cancer is unknown. We report on the largest single-institution experience using adjuvant high-dose-rate vaginal brachytherapy (VBT) for surgically staged women with FIGO stage I UPSC or CC endometrial cancer. Methods and Materials: From 1998-2011, 103 women with FIGO 2009 stage I UPSC (n=74), CC (n=21), or mixed UPSC/CC (n=8) endometrial cancer underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy followed by adjuvant high-dose-rate VBT. Nearly all patients (n=98, 95%) also underwent extended lymph node dissection of pelvic and paraortic lymph nodes. All VBT was performed with a vaginal cylinder, treating to a dose of 2100 cGy in 3 fractions. Thirty-five patients (34%) also received adjuvant chemotherapy. Results: At a median follow-up time of 36 months (range, 1-146 months), 2 patients had experienced vaginal recurrence, and the 5-year Kaplan Meier estimate of vaginal recurrence was 3%. The rates of isolated pelvic recurrence, locoregional recurrence (vaginal + pelvic), and extrapelvic recurrence (including intraabdominal) were similarly low, with 5-year Kaplan-Meier estimates of 4%, 7%, and 10%, respectively. The estimated 5-year overall survival was 84%. On univariate analysis, delivery of chemotherapy did not affect recurrence or survival. Conclusions: VBT is effective at preventing vaginal relapse in women with surgical stage I UPSC or CC endometrial cancer. In this cohort of patients who underwent comprehensive surgical staging, the risk of isolated pelvic or extrapelvic relapse was low, implying that more extensive adjuvant radiation therapy is likely unnecessary.

  5. Retinoic acid promotes primary fetal alveolar epithelial type II cell proliferation and differentiation to alveolar epithelial type I cells.

    PubMed

    Gao, Rui-wei; Kong, Xiang-yong; Zhu, Xiao-xi; Zhu, Guo-qing; Ma, Jin-shuai; Liu, Xiu-xiang

    2015-05-01

    Retinoic acid (RA) plays an important role in lung development and maturation. Many stimuli can induce alveolar epithelial cell damage which will result in the injury of lung parenchyma. The aim of this study was to observe the effect of RA on the proliferation and differentiation of primary fetal alveolar epithelial type II cells (fAECIIs). Primary fAECIIs were isolated from fetal rats at 19 d of gestation and purified by a differential centrifugation and adhesion method. The cells were randomly divided into control (dimethyl sulfoxide, DMSO) and RA groups. Cell proliferation, viability, apoptosis, cycle, and expression of target protein were examined at 24, 48, and 72 h. We found that the proliferation and viability of cells in the RA-exposed group significantly increased compared with the DMSO control group. The proportion (%) of cells in the G2 and S phases in the RA group was significantly higher than that in control group cells. The proportion (%) of both early apoptotic cells and late apoptotic cells decreased significantly in cells exposed to RA compared with cells exposed to DMSO. RA significantly enhanced the expression of aquaporin 5 (AQP5). The expression level of pulmonary surfactant C (SPC) was elevated after cells were exposed to RA for 24 and 72 h but was inhibited when cells were exposed to RA for 48 h. These results suggest that RA promotes fAECII proliferation by improving cell viability, promoting S phase entry and inhibiting apoptosis and RA promotes fAECIIs differentiation to alveolar epithelial type I cells (AECIs). PMID:25515249

  6. Primary epithelial cell models for cystic fibrosis research.

    PubMed

    Randell, Scott H; Fulcher, M Leslie; O'Neal, Wanda; Olsen, John C

    2011-01-01

    When primary human airway epithelial (hAE) cells are grown in vitro on porous supports at an air-liquid interface (ALI), they recapitulate in vivo morphology and key physiologic processes. These cultures are useful for studying respiratory tract biology and diseases and for testing new cystic fibrosis (CF) therapies. This chapter gives protocols enabling creation of well-differentiated primary CF and non-CF airway epithelial cell cultures with non-proprietary reagents. We also discuss the production of retroviral and lentiviral vectors, the derivation of hAE cell lines, reporter gene assays, and the evolving science of gene overexpression and knockdown in ALI hAE cultures. PMID:21547740

  7. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  8. Medullary thymic epithelial cell depletion leads to autoimmune hepatitis

    PubMed Central

    Bonito, Anthony J.; Aloman, Costica; Fiel, M. Isabel; Danzl, Nichole M.; Cha, Sungwon; Weinstein, Erica G.; Jeong, Seihwan; Choi, Yongwon; Walsh, Matthew C.; Alexandropoulos, Konstantina

    2013-01-01

    TRAF6, an E3 ubiquitin protein ligase, plays a critical role in T cell tolerance by regulating medullary thymic epithelial cell (mTEC) development. mTECs regulate T cell tolerance by ectopically expressing self-antigens and eliminating autoreactive T cells in the thymus. Here we show that mice with mTEC depletion due to conditional deletion of Traf6 expression in murine thymic epithelial cells (Traf6?TEC mice) showed a surprisingly narrow spectrum of autoimmunity affecting the liver. The liver inflammation in Traf6?TEC mice exhibited all the histological and immunological characteristics of human autoimmune hepatitis (AIH). The role of T cells in AIH establishment was supported by intrahepatic T cell population changes and AIH development after transfer of liver T cells into immunodeficient mice. Despite a 50% reduction in natural Treg thymic output, peripheral tolerance in Traf6?TEC mice was normal, whereas compensatory T regulatory mechanisms were evident in the liver of these animals. These data indicate that mTECs exert a cell-autonomous role in central T cell tolerance and organ-specific autoimmunity, but play a redundant role in peripheral tolerance. These findings also demonstrate that Traf6?TEC mice are a relevant model with which to study the pathophysiology of AIH, as well as autoantigen-specific T cell responses and regulatory mechanisms underlying this disease. PMID:23867620

  9. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  10. Nucleotide exchange factor ECT2 regulates epithelial cell polarity.

    PubMed

    Liu, Xiu Fen; Ohno, Shigeo; Miki, Toru

    2006-10-01

    Cell polarity regulates diverse biological events such as localization of embryonic determinants and establishment of tissue and organ architecture. Epithelial cell polarity is regulated by the polarity complex Par6/Par3/atypical protein kinase C (aPKC). We previously found that the nucleotide exchange factor ECT2 associates with this polarity complex and regulates aPKC activity, but the role of ECT2 in cell polarity is still unclear. Here we show that expression of a dominant negative (ECT2-N2) or constitutively active (ECT2-DeltaN5) form of ECT2 inhibits normal cyst formation of MDCK cells in 3-dimensional collagen gels. Central lumens were not observed in cysts formed by cells expressing either ECT2-DeltaN5 or ECT2-N2. Apical localization of ZO-1 and basolateral localization of beta-catenin were no longer observed in these cells. Interestingly, cells expressing ECT2-N2 did form normal cysts when cultured in the basement membrane matrix Matrigel instead of collagen gels. Addition of a major Matrigel component, laminin, partially rescued the normal cyst formation inhibited by ECT2-N2 in 3-dimensional collagen gels. Thus, signaling through laminin might override the defects of signaling through collagen and ECT2. Whereas ECT2-N2 inhibited the lumen formation of MDCK cysts, caspase-3, which is reportedly involved in lumen formation through apoptosis, was activated at various locations of cells in the cysts. It is likely that perturbation of ECT2 signaling inhibits the establishment of epithelial cell polarity leading to the inhibition of selected elimination of cells at the center of cysts. Thus, ECT2 appears to play a critical role in epithelial cell polarity. PMID:16495035

  11. Characterization of butyrate uptake by nontransformed intestinal epithelial cell lines.

    PubMed

    Gonçalves, Pedro; Araújo, João R; Martel, Fátima

    2011-03-01

    Butyrate (BT) is one of the main end products of anaerobic bacterial fermentation of dietary fiber within the human colon. Among its recognized effects, BT inhibits colon carcinogenesis. Our aim was to characterize uptake of BT by two nontransformed intestinal epithelial cell lines: rat small intestinal epithelial (IEC-6) and fetal human colonic epithelial (FHC) cells. Uptake of ¹⁴C-BT by IEC-6 cells was (1) time- and concentration-dependent; (2) pH-dependent; (3) Na+-, Cl⁻- and energy-dependent; (4) inhibited by BT structural analogues; (5) sensitive to monocarboxylate transporter 1 (MCT1) inhibitors; and (6) insensitive to DIDS and amiloride. IEC-6 cells express both MCT1 and Na+-coupled monocarboxylate transporter 1 (SMCT1) mRNA. We conclude that ¹⁴C-BT uptake by IEC-6 cells mainly involves MCT1, with a small contribution of SMCT1. Acute exposure to ethanol, acetaldehyde, indomethacin, resveratrol and quercetin reduced ¹⁴C-BT uptake. Chronic exposure to resveratrol and quercetin reduced ¹⁴C-BT uptake but had no effect on either MCT1 or SMCT1 mRNA levels. Uptake of ¹⁴C-BT by FHC cells was time- and concentration-dependent but pH-, Na+-, Cl⁻- and energy-independent and insensitive to BT structural analogues and MCT1 inhibitors. Although MCT1 (but not SMCT1) mRNA expression was found in FHC cells, the characteristics of ¹⁴C-BT uptake by FHC cells did not support either MCT1 or SMCT1 involvement. In conclusion, uptake characteristics of ¹⁴C-BT differ between IEC-6 and FHC cells. IEC-6 cells demonstrate MCT1- and SMCT1-mediated transport, while FHC cells do not. PMID:21286694

  12. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells.

    PubMed

    Huang, Sarah X L; Islam, Mohammad Naimul; O'Neill, John; Hu, Zheng; Yang, Yong-Guang; Chen, Ya-Wen; Mumau, Melanie; Green, Michael D; Vunjak-Novakovic, Gordana; Bhattacharya, Jahar; Snoeck, Hans-Willem

    2014-01-01

    The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP-agonists to signaling pathways critical for early lung development in the mouse-recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells. PMID:24291815

  13. Inhibition of autophagy in peripheral blood mononuclear cells by vaginal fluid from women with a malignant adnexal mass.

    PubMed

    Orfanelli, Theofano; Doulaveris, Georgios; Holcomb, Kevin; Jeong, Jiyeon M; Sisti, Giovanni; Kanninen, Tomi T; Caputo, Thomas A; Gupta, Divya; Witkin, Steven S

    2015-12-15

    Inhibition of autophagy is a characteristic of ovarian cancer. We determined whether inhibition of autophagy by vaginal fluid could provide a non-invasive test for cancer risk stratification in women presenting with an adnexal mass. Vaginal fluid supernatants from 90 women undergoing evaluation for a suspicious adnexal mass were incubated with peripheral blood mononuclear cells (PBMCs) obtained from healthy women under conditions that induce autophagy. Rapamycin, an autophagy inducer, was added to some cultures. After 48 hr the cells were collected, lysed and assayed by ELISA for intracellular p62 concentration. p62 is a cytoplasmic protein that is consumed during autophagy induction. Its concentration is inversely proportional to the extent of autophagy induction. Clinical information including pathological diagnoses was obtained after completion of laboratory studies. Mean p62 levels were 9.4 ng/ml in the 21 women with a subsequent malignant diagnosis, 4.5 ng/ml in the eight women with a borderline tumor diagnosis and 3.6 ng/ml in the 61 women with benign disease (p?vaginal fluid-PBMC co-incubation, p62 levels in samples from women with a malignant diagnosis decreased to 3.3 ng/ml, a level comparable to what was observed with the nonmalignant samples. Vaginal fluid inhibition of autophagy can differentiate between women with malignant and benign adnexal masses. PMID:26132572

  14. EpithelialMesenchymal Transition in Kidney Tubular Epithelial Cells Induced by Globotriaosylsphingosine and Globotriaosylceramide

    PubMed Central

    Jeon, Yeo Jin; Jung, Namhee; Park, Joo-Won; Park, Hae-Young; Jung, Sung-Chul

    2015-01-01

    Fabry disease is a lysosomal storage disorder caused by deficiency of alpha-galactosidase A (?-gal A), which results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. Globotriaosylsphingosine (lyso-Gb3), a deacylated Gb3, is also increased in the plasma of patients with Fabry disease. Renal fibrosis is a key feature of advanced Fabry disease patients. Therefore, we evaluated the association of Gb3 and lyso-Gb3 accumulation and the epithelialmesenchymal transition (EMT) on tubular epithelial cells of the kidney. In HK2 cells, exogenous treatments of Gb3 and lyso-Gb3 increased the expression of TGF-?, EMT markers (N-cadherin and ?-SMA), and phosphorylation of PI3K/AKT, and decreased the expression of E-cadherin. Lyso-Gb3, rather than Gb3, strongly induced EMT in HK2 cells. In the mouse renal mesangial cell line, SV40 MES 13 cells, Gb3 strongly induced phenotype changes. The EMT induced by Gb3 was inhibited by enzyme ?-gal A treatment, but EMT induced by lyso-Gb3 was not abrogated by enzyme treatment. However, TGF-? receptor inhibitor (TRI, SB525334) inhibited the activation of TGF-? and EMT markers in HK2 cells with Gb3 and lyso-Gb3 treatments. This study suggested that increased plasma lyso-Gb3 has a crucial role in the development of renal fibrosis through the cell-specific induction of the EMT in Fabry disease, and that TRI treatment, alongside enzyme replacement therapy, could be a potential therapeutic option for patients with Fabry disease. PMID:26291612

  15. Importance of the keratinized epithelial cell in bacterial adherence.

    PubMed

    Bibel, D J; Aly, R; Shinefield, H R; Maibach, H I; Strauss, W G

    1982-10-01

    The effect of cellular pathology and keratinization of skin and nasal cells upon binding of Staphylococcus aureus were examined. Adherence with epithelial cells obtained from either the skin or nasal mucosa of patients with atopic dermatitis was greater than that observed with normal cells (p less than 0.001); the difference in adherence between psoriatic and normal cells was not statistically significant. Tested nasal cells were microscopically differentiated into 4 general types based on stage or layer of keratinization: spinous, low granular, high granular, and keratin. The degree of adherence was related to the progress of keratinization. Data indicated the existence of 2 types of receptors for S. aureus on nasal cells: One, present upon both granular and fully keratinized cells, is not blocked by teichoic acid and appears responsible for the higher bacterial counts on atopic cells; the second is found on keratinized cells only and is susceptible to teichoic acid. PMID:6182249

  16. Normal and tumour cervical cells respond differently to vaginal lactobacilli, independent of pH and lactate.

    PubMed

    Motevaseli, Elahe; Shirzad, Mahdieh; Akrami, Seyed Mohammad; Mousavi, Azam-Sadat; Mirsalehian, Akbar; Modarressi, Mohammad Hossein

    2013-07-01

    Cervical cancer is a human papilloma virus (HPV)-related cancer, but most HPV infections are transient or intermittent and resolve spontaneously. Thus, other factors, such as cervical microflora, which are dominated by lactobacilli, must be involved in invasive cervical carcinoma development after HPV infection. Previous studies have demonstrated that lactobacilli have antitumour effects, and it is possible that vaginal lactobacilli prevent cervical cancer. Here we examined the proliferative and apoptotic responses of normal and tumour cervical cells to common vaginal lactobacilli components by investigating human normal fibroblast-like cervical (normal cervical) and HeLa (cervical tumour) cell responses to Lactobacillus gasseri and Lactobacillus crispatus. The effects of different lactobacilli components, such as culture supernatants, cytoplasmic extracts, cell-wall extracts and live cells, were determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, trypan blue staining, lactate dehydrogenase assay and colorimetric caspase-3 activity assay. Changes in caspase-3 and human chorionic gonadotropin β (hCGβ) expression were analysed by quantitative RT-PCR. Tumour cell growth inhibition by culture supernatants was higher than that by pH- and lactate-adjusted controls. However, the effects of the supernatants on normal cells were similar to those of lactate-adjusted controls. Apoptosis was inhibited by supernatants, which was consistent with higher hCGβ expression since hCG inhibits apoptosis. Our study demonstrated that common vaginal lactobacilli exert cytotoxic effects on cervical tumour cells, but not on normal cells, and that this cytotoxicity is independent of pH and lactate. Our results encourage further studies on the interaction between lactobacilli and cervical cells, and administration of common vaginal lactobacilli as probiotics. PMID:23618799

  17. CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

  18. Epstein-Barr Virus Transcytosis through Polarized Oral Epithelial Cells

    PubMed Central

    Herrera, Rossana; Palefsky, Joel M.

    2013-01-01

    Although Epstein-Barr virus (EBV) is an orally transmitted virus, viral transmission through the oropharyngeal mucosal epithelium is not well understood. In this study, we investigated how EBV traverses polarized human oral epithelial cells without causing productive infection. We found that EBV may be transcytosed through oral epithelial cells bidirectionally, from both the apical to the basolateral membranes and the basolateral to the apical membranes. Apical to basolateral EBV transcytosis was substantially reduced by amiloride, an inhibitor of macropinocytosis. Electron microscopy showed that virions were surrounded by apical surface protrusions and that virus was present in subapical vesicles. Inactivation of signaling molecules critical for macropinocytosis, including phosphatidylinositol 3-kinases, myosin light-chain kinase, Ras-related C3 botulinum toxin substrate 1, p21-activated kinase 1, ADP-ribosylation factor 6, and cell division control protein 42 homolog, led to significant reduction in EBV apical to basolateral transcytosis. In contrast, basolateral to apical EBV transcytosis was substantially reduced by nystatin, an inhibitor of caveolin-mediated virus entry. Caveolae were detected in the basolateral membranes of polarized human oral epithelial cells, and virions were detected in caveosome-like endosomes. Methyl β-cyclodextrin, an inhibitor of caveola formation, reduced EBV basolateral entry. EBV virions transcytosed in either direction were able to infect B lymphocytes. Together, these data show that EBV transmigrates across oral epithelial cells by (i) apical to basolateral transcytosis, potentially contributing to initial EBV penetration that leads to systemic infection, and (ii) basolateral to apical transcytosis, which may enable EBV secretion into saliva in EBV-infected individuals. PMID:23698302

  19. Vitamin C inhibit the proliferation, migration and epithelial-mesenchymal-transition of lens epithelial cells by destabilizing HIF-1?

    PubMed Central

    Zhao, Lin; Quan, Yanlong; Wang, Jianming; Wang, Feng; Zheng, Yuping; Zhou, Aiyi

    2015-01-01

    Posterior capsular opacification (PCO), the main complication of cataract surgery, is mainly caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the residual lens epithelial cells (LECs).Vitamin C was reported to reduce the risk of forming a cataract. However, there has been no study showing the association between vitamin C and PCO. In this study, we found that vitamin C could inhibit the migration and proliferation of human lens epithelial cells. We also found that vitamin C could increase the proline hydroxylation of HIF-1? and reduce the activity of HIF-1?. Moreover, vitamin C could not inhibit the activity of proline-mutant HIF-1? (402/564). Overexpression of wild-type HIF-1? or proline-mutant HIF-1? was found to increase the proliferation and migration of human lens epithelial cells. Differently, vitamin C could inhibit the proliferation and migration in wild-type HIF-1?-overexpressing lens epithelial cells but not the proline-mutant HIF-1?-overexpressing cells. Additionally, vitamin C was also found to inhibit the expression of EMT transcription factors TWIST. We then found that vitamin C could repress the EMT phenotypes induced by the overexpression of wild-type HIF-1? but not the proline-mutant HIF-1?. These results provide evidence that vitamin C plays a role in the repression of proliferation, migration, and EMT of human lens epithelial cells by destabilizing HIF-1?. PMID:26628999

  20. Photodynamic treatment of lens epithelial cells for cataract surgery

    NASA Astrophysics Data System (ADS)

    Lingua, Robert W.; Parel, Jean-Marie A.; Simon, Gabriel; Li, Kam

    1991-06-01

    Photodynamic therapy (PDT) eiiploying Dihematopor*iyrin ethers (DHE) (Photofrin II) at pharmacologic lvels, has been denonstrate3 to kill rabbit lens epithelial cells, in vivo. This in vitro study, reports on the minimal necessary parameters for rabbit lens epithelial cell death. Explants of rabbit lenses were incubated in various concentrations of DHE (1O,, 100, 500, 1000 ug/ml) for 1, 2, or 5 minutes. 30 to 120 Joules/an of collimated 514.5 nm Argon laser light re delivered to the locier concentrations of 10, 50, and 100 ug,'ml DHE treated cells. One hundre1 fifteen explants were treated, in all. Higher concentrations of DHE alone (500 and 1000 ug/ml) were sufficient to induce cellular swelling. Lower concentrations required light for cellular effect. Trypan blue staining revealed cell death at these minimal pa9ieters: DHE 50 ug/ml, incubation 1 minute, 514.5 r Argon light 1.0 Watt/an for 30 sec (30 Joules) . In future studies, these rameters will be tested in vivo, for their ability to eliminate lens epithelial proliferation after cataract surgery.

  1. Serratia marcescens is injurious to intestinal epithelial cells.

    PubMed

    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

  2. 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

  3. Elevated tropomyosin expression is associated with epithelialmesenchymal transition of lens epithelial cells

    PubMed Central

    Kubo, Eri; Hasanova, Nailia; Fatma, Nigar; Sasaki, Hiroshi; Singh, Dhirendra P

    2013-01-01

    Injury to lens epithelial cells (LECs) leads to epithelialmesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1?/2? suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of ?-smooth muscle actin (?-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1?/2? following EMT/PCO formation. Expression of Tpm1?/2? was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2? mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1?/2?, as evidenced by increased expression of ?-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis. PMID:23205574

  4. Elevated tropomyosin expression is associated with epithelial-mesenchymal transition of lens epithelial cells.

    PubMed

    Kubo, Eri; Hasanova, Nailia; Fatma, Nigar; Sasaki, Hiroshi; Singh, Dhirendra P

    2013-01-01

    Injury to lens epithelial cells (LECs) leads to epithelial-mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1?/2? suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of ?-smooth muscle actin (?-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1?/2? following EMT/PCO formation. Expression of Tpm1?/2? was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2? mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1?/2?, as evidenced by increased expression of ?-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis. PMID:23205574

  5. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis.

    PubMed

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  6. Left–right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    PubMed Central

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left–right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left–right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left–right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  7. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3?, and the p85? subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3? pathway. PMID:26360608

  8. *Iron accumulation in bronchial epithelial cells is dependent on concurrent sodium transport

    EPA Science Inventory

    Airway epithelial cells prevent damaging effects of extracellular iron by taking up the metal and sequestering it within intracellular ferritin. Epithelial iron transport is associated with transcellular movement of other cations including changes in the expression or activity of...

  9. A molecular mechanotransduction pathway regulates collective migration of epithelial cells.

    PubMed

    Das, Tamal; Safferling, Kai; Rausch, Sebastian; Grabe, Niels; Boehm, Heike; Spatz, Joachim P

    2015-03-01

    Collective movement of epithelial cells drives essential multicellular organization during various fundamental physiological processes encompassing embryonic morphogenesis, cancer and wound healing. Yet the molecular mechanism that ensures the coordinated movement of many cells remains elusive. Here we show that a tumour suppressor protein, merlin, coordinates collective migration of tens of cells, by acting as a mechanochemical transducer. In a stationary epithelial monolayer and also in three-dimensional human skin, merlin localizes to cortical cell-cell junctions. During migration initiation, a fraction of cortical merlin relocalizes to the cytoplasm. This relocalization is triggered by the intercellular pulling force of the leading cell and depends on the actomyosin-based cell contractility. Then in migrating cells, taking its cue from the intercellular pulling forces, which show long-distance ordering, merlin coordinates polarized Rac1 activation and lamellipodium formation on the multicellular length scale. Together, these results provide a distinct molecular mechanism linking intercellular forces to collective cell movements in migrating epithelia. PMID:25706233

  10. Polarized angular dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures.

    PubMed

    Mourant, J R; Johnson, T M; Carpenter, S; Guerra, A; Aida, T; Freyer, J P

    2002-07-01

    An understanding of the relationship between tissue structures and light scattering from tissue will help facilitate the development and acceptance of noninvasive optical diagnostics including elastic scattering spectroscopy, diffuse reflectance, and optical coherence tomography. For example, a quantitative model of the structures that scatter light in epithelial cells would allow determination of what structures control the characteristics of in vivo light transport measurements and subsequently could provide a detailed relationship between cellular structures and optical measurements. We have determined the size distribution of refractive index structure variations in epithelial cells as well as in nuclei isolated from epithelial cells from measurements of the angular dependence of polarized light scattering. The quantitative size distributions we obtained for both whole cells and isolated nuclei include particles with effective radii of 2 microm to 10 nm or less and contain orders of magnitude more small particles than large particles. These results demonstrate that not only are biological cells very heterogeneous, but so are the nuclei within them. Light scattering is likely sensitive to structures smaller than those commonly investigated by standard pathology methods. PMID:12175287

  11. Epithelial Cell Polarity Determinant CRB3 in Cancer Development

    PubMed Central

    Li, Pingping; Mao, Xiaona; Ren, Yu; Liu, Peijun

    2015-01-01

    Cell polarity, which is defined as asymmetry in cell shape, organelle distribution and cell function, is essential in numerous biological processes, including cell growth, cell migration and invasion, molecular transport, and cell fate. Epithelial cell polarity is mainly regulated by three conserved polarity protein complexes, the Crumbs (CRB) complex, partitioning defective (PAR) complex and Scribble (SCRIB) complex. Research evidence has indicated that dysregulation of cell polarity proteins may play an important role in cancer development. Crumbs homolog 3 (CRB3), a member of the CRB complex, may act as a cancer suppressor in mouse kidney epithelium and mouse mammary epithelium. In this review, we focus on the current data available on the roles of CRB3 in cancer development. PMID:25552927

  12. Isolation of intestinal epithelial cells and evaluation of transport functions

    SciTech Connect

    Kimmich, G.A.

    1990-01-01

    Epithelial cells can be isolated from the small intestine of chickens by a procedure involving hyaluronidase treatment of the intact tissue. The isolated cells retain a high degree of functional activity as assessed by the formation of 70-fold gradients of alpha-MG. Stability of the sugar gradients reflects maintenance of stable electrochemical Na+ gradients across the plasma membrane. The cells can be used to evaluate the properties of Na(+)-dependent sugar transport, Na(+)-independent sugar transport, ion transport, metabolism, membrane potentials, and the integration of these events, all of which are important to achieving a stable sugar gradient.

  13. The yin and yang of intestinal epithelial cells in controlling dendritic cell function

    PubMed Central

    Iliev, Iliyan D.; Matteoli, Gianluca; Rescigno, Maria

    2007-01-01

    Recent work suggests that dendritic cells (DCs) in mucosal tissues are educated by intestinal epithelial cells (IECs) to suppress inflammation and promote immunological tolerance. After attack by pathogenic microorganisms, however, non-educated DCs are recruited from nearby areas, such as the dome of Peyer's patches (PPs) and the blood, to initiate inflammation and the ensuing immune response to the invader. Differential epithelial cell (EC) responses to commensals and pathogens may control these two tolorogenic and immunogenic functions of DCs. PMID:17893197

  14. Human corneal epithelial cell response to substrate stiffness.

    PubMed

    Molladavoodi, Sara; Kwon, Hyock-Ju; Medley, John; Gorbet, Maud

    2015-01-01

    It has been reported that mechanical stimulus can affect cellular behavior. While induced differentiation in stem cells and proliferation and directional migration in fibroblasts are reported as responses to mechanical stimuli, little is known about the response of cells from the cornea. In the present study, we investigated whether changes in substrate stiffness (measured by elastic modulus) affected the behavior of human corneal epithelial cells (HCECs). Polyacrylamide substrates with different elastic moduli (compliant, medium and stiff) were prepared and HCECs were cultured on them. HCECs responses, including cell viability, apoptosis, intercellular adhesion molecule-1 (ICAM-1) expression, integrin-?3?1 expression and changes in cytoskeleton structure (actin fibers) and migratory behavior, were studied. No statistically significant cell activation, as measured by ICAM-1 expression, was observed. However, on compliant substrates, a higher number of cells were found to be apoptotic and disrupted actin fibers were observed. Furthermore, cells displayed a statistically significant lower migration speed on compliant substrates when compared with the stiffer substrates. Thus, corneal epithelial cells respond to changes in substrate stiffness, which may have implications in the understanding and perhaps treatment of corneal diseases, such as keratoconus. PMID:25305512

  15. Hyperoxia alters the mechanical properties of alveolar epithelial cells

    PubMed Central

    Roan, Esra; Wilhelm, Kristina; Bada, Alex; Makena, Patrudu S.; Gorantla, Vijay K.; Sinclair, Scott E.

    2012-01-01

    Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability. PMID:22467640

  16. Human alveolar epithelial type II cells in primary culture

    PubMed Central

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-01-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (?ENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, ?ENaC, and aquaporin (AQP)-3, which was maintained over 20days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-?. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  17. TCDD alters medial epithelial cell differentiation during palatogenesis

    SciTech Connect

    Abbott, B.D.; Birnbaum, L.S. )

    1989-06-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism is the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of (3H)TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation.

  18. Clinical Features of Bacterial Vaginosis in a Murine Model of Vaginal Infection with Gardnerella vaginalis

    PubMed Central

    Gilbert, Nicole M.; Lewis, Warren G.; Lewis, Amanda L.

    2013-01-01

    Bacterial vaginosis (BV) is a dysbiosis of the vaginal flora characterized by a shift from a Lactobacillus-dominant environment to a polymicrobial mixture including Actinobacteria and Gram-negative bacilli. BV is a common vaginal condition in women and is associated with increased risk of sexually transmitted infection and adverse pregnancy outcomes such as preterm birth. Gardnerella vaginalis is one of the most frequently isolated bacterial species in BV. However, there has been much debate in the literature concerning the contribution of G. vaginalis to the etiology of BV, since it is also present in a significant proportion of healthy women. Here we present a new murine vaginal infection model with a clinical isolate of G. vaginalis. Our data demonstrate that this model displays key features used clinically to diagnose BV, including the presence of sialidase activity and exfoliated epithelial cells with adherent bacteria (reminiscent of clue cells). G. vaginalis was capable of ascending uterine infection, which correlated with the degree of vaginal infection and level of vaginal sialidase activity. The host response to G. vaginalis infection was characterized by robust vaginal epithelial cell exfoliation in the absence of histological inflammation. Our analyses of clinical specimens from women with BV revealed a measureable epithelial exfoliation response compared to women with normal flora, a phenotype that, to our knowledge, is measured here for the first time. The results of this study demonstrate that G. vaginalis is sufficient to cause BV phenotypes and suggest that this organism may contribute to BV etiology and associated complications. This is the first time vaginal infection by a BV associated bacterium in an animal has been shown to parallel the human disease with regard to clinical diagnostic features. Future studies with this model should facilitate investigation of important questions regarding BV etiology, pathogenesis and associated complications. PMID:23527214

  19. Elastic properties of epithelial cells probed by atomic force microscopy.

    PubMed

    Brckner, Bastian R; Janshoff, Andreas

    2015-11-01

    Cellular mechanics plays a crucial role in many biological processes such as cell migration, cell growth, embryogenesis, and oncogenesis. Epithelia respond to environmental cues comprising biochemical and physical stimuli through defined changes in cell elasticity. For instance, cells can differentiate between certain properties such as viscoelasticity or topography of substrates by adapting their own elasticity and shape. A living cell is a complex viscoelastic body that not only exhibits a shell architecture composed of a membrane attached to a cytoskeleton cortex but also generates contractile forces through its actomyosin network. Here we review cellular mechanics of single cells in the context of epithelial cell layers responding to chemical and physical stimuli. This article is part of a Special Issue entitled: Mechanobiology. PMID:26193077

  20. Airway epithelial cells from asthmatic children differentially express proremodeling factors

    PubMed Central

    Lopez-Guisa, Jesus M.; Powers, Claire; File, Daniele; Cochrane, Elizabeth; Jimenez, Nathalia; Debley, Jason S.

    2012-01-01

    Background The airway epithelium can express factors that drive subepithelial airway remodeling. TGF-β2, vascular epithelial growth factor (VEGF), a disintegrin and metalloprotease 33 (ADAM33), and periostin are hypothesized to be involved in subepithelial remodeling and are overexpressed in adult asthmatic airways. Epidemiologic data suggest that lung function deficits in asthmatic patients are acquired in childhood. Objectives We sought to determine whether airway epithelial cells (AECs) from asthmatic children differentially express TGF-β2, VEGF, ADAM33, or periostin compared with cells from atopic nonasthmatic and healthy children intrinsically or in response to IL-4/IL-13 stimulation. Methods Bronchial and nasal epithelial cells were obtained from brushings from well-characterized asthmatic (n = 16), atopic nonasthmatic (n = 9), and healthy (n = 15) children after achievement of anesthesia for elective procedures. After differentiation at an air-liquid interface (ALI) for 3 weeks, conditioned media were sampled and RNA was extracted from unstimulated and IL-4/IL-13–stimulated cultures. TGF-β2 and VEGF levels were measured with ELISA. ADAM33 and periostin expression was assessed by using real-time PCR. Results TGF-β2 and VEGF production was significantly greater in bronchial and nasal ALI cultures from asthmatic children than in cultures from atopic nonasthmatic and healthy children. TGF-β2 levels increased significantly in asthmatic cultures after IL-4/IL-13 stimulation. Within-subject correlation between nasal and bronchial ALI production of TGF-β2 (r = 0.64, P = .001) and VEGF (r = 0.73, P < .001) was good. Periostin expression was 3.7-fold higher in bronchial cells (P < .001) and 3.9-fold higher in nasal cells (P < .004) from asthmatic children than in cells from atopic nonasthmatic or healthy children. ADAM33 was not differentially expressed by AECs from asthmatic patients compared with that from cells from atopic nonasthmatic or healthy children. Conclusion AECs from asthmatic children differentially express TGF-β2, VEGF, and periostin compared with cells from atopic nonasthmatic and healthy children. Nasal epithelial cells might be a suitable surrogate for bronchial cells that could facilitate investigation of the airway epithelium in future longitudinal pediatric studies. PMID:22227417

  1. Adherent properties of Helicobacter pylori to human epithelial cells

    PubMed Central

    Wang, Zheng-Xiang; Shen, Hou-Feng; Chen, Hong-Ju

    1997-01-01

    AIM: To study the properties and factors of Helicobacter pylori (H. pylori) adherence to human epithelial cells. METHODS:The adherent properties of human epithelial cells were studied using a group of isolated H. pylori strains, anti-H. pylori monoclonal antibodies and varied pH environment in in vitro adherence model with HEp2 cells. RESULTS: H. pylori YC 11A was able to adhere to HEp2 cells specifically and its adherence efficiency reached the highest (81%) within 3 h after incubation with HEp2 cells. There was no significant difference between adherence in air and in 5% oxygen. The monoclonal antibodies specific to H. pylori predominant antigens did not inhibit activities on adherence of H. pylori to HEp2 cells. The pH value significantly affected the adherence process and the optimal pH was 3.0-4.6. CONCLUSION: H. pylori specifically adheres to HEp2 cells, and pH value significantly affects this process. A high level of anti-H. pylori predominant antibodies in serum may have no protective activities against H. pylori infection.

  2. Protein-Coated Nanoparticles Are Internalized by the Epithelial Cells of the Female Reproductive Tract and Induce Systemic and Mucosal Immune Responses

    PubMed Central

    Howe, Savannah E.; Konjufca, Vjollca H.

    2014-01-01

    The female reproductive tract (FRT) includes the oviducts (fallopian tubes), uterus, cervix and vagina. A layer of columnar epithelium separates the endocervix and uterus from the outside environment, while the vagina is lined with stratified squamous epithelium. The mucosa of the FRT is exposed to antigens originating from microflora, and occasionally from infectious microorganisms. Whether epithelial cells (ECs) of the FRT take up (sample) the lumen antigens is not known. To address this question, we examined the uptake of 20–40 nm nanoparticles (NPs) applied vaginally to mice which were not treated with hormones, epithelial disruptors, or adjuvants. We found that 20 and 40 nm NPs are quickly internalized by ECs of the upper FRT and within one hour could be observed in the lymphatic ducts that drain the FRT, as well as in the ileac lymph nodes (ILNs) and the mesenteric lymph nodes (MLNs). Chicken ovalbumin (Ova) conjugated to 20 nm NPs (NP-Ova) when administered vaginally reaches the internal milieu in an immunologically relevant form; thus vaginal immunization of mice with NP-Ova induces systemic IgG to Ova antigen. Most importantly, vaginal immunization primes the intestinal mucosa for secretion of sIgA. Sub-cutaneous (s.c) boosting immunization with Ova in complete Freund's adjuvant (CFA) further elevates the systemic (IgG1 and IgG2c) as well as mucosal (IgG1 and sIgA) antibody titers. These findings suggest that the modes of antigen uptake at mucosal surfaces and pathways of antigen transport are more complex than previously appreciated. PMID:25490456

  3. Viability of freeze dried microencapsulated human retinal pigment epithelial cells.

    PubMed

    Wikstrm, Jonna; Elomaa, Matti; Nevala, Laura; Rikknen, Johanna; Heljo, Petteri; Urtti, Arto; Yliperttula, Marjo

    2012-09-29

    Encapsulated human retinal pigment epithelial cell line ARPE-19 has been successfully used in experimental cell therapy of retinal degenerations and Parkinson's disease, but the long-term storage of encapsulated cells is still an unresolved question. Reconstitution of viable encapsulated cells from dry form would benefit the development of cell therapy products. We freeze dried and reconstituted microencapsulated ARPE19 and ARPE19-SEAP cells. Cross-linked alginate matrix with polycation (poly-l-lysine, cationic starch) coating was used for microencapsulation. Cell viability was assessed with fluorescence microscopy and oxygen consumption of the cells. Freeze dried and reconstituted cell microcapsules were imaged using scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM). We show partial viability of microencapsulated cells after freeze-drying. Unlike poly-l-lysine (PLL) coating, cationic starch supported microcapsule shape and cell viability during freeze-drying. Trehalose pre-treatment augmented cell viability. Likewise, some lyoprotectants (trehalose, glycerol) enabled preservation of cell viability. Upon reconstitution the freeze dried cell microcapsules rapidly regained their original spherical shape. This proof-of-concept study demonstrates that microencapsulated cells can retain their viability during freeze-drying. Therefore, this approach can be further optimized for the benefit of cell therapy product development. PMID:22820032

  4. Cooperation between epithelial cells demonstrated by potassium transfer

    SciTech Connect

    Ledbetter, M.L.; Young, G.J.; Wright, E.R.

    1986-02-01

    Junction-mediated communication can be measured in fibroblast cultures by determining the ability of mixed cultures of cells sensitive and resistant to ouabain to concentrate K+ in the presence of ouabain. We now report the extension of this assay procedure to cultured epithelial cells. Hamster kidney (HaK) cells maintain their ability to concentrate K+ in ouabain at levels inhibitory to dog kidney (MDCK) cells. When HaK and MDCK cells were cultured together in ouabain-containing medium, the K+ (measured as 86Rb+) in the mixed population was greater than expected if the cells were not interacting. The degree of enhancement, expressed as index of cooperation, depended on the numbers of cells in the cultures, their opportunity for cell-to-cell contact, and (above a certain permissive level) the concentration of ouabain. As with other cell types, protein synthesis in MDCK cells depends on maintenance of cell K+. Autoradiography of cells incubated with (3H)leucine demonstrated that MDCK cells in ouabain-treated mixed cultures were able to synthesize proteins only when physically adjacent to HaK cells. The transmission of labeled nucleosides among the cells provides independent evidence of the phenomenon of cooperation, probably mediated by gap junctions. This system offers promise for investigation of stimuli modulating junctional communication.

  5. Native type IV collagen induces an epithelial to mesenchymal transition-like process in mammary epithelial cells MCF10A.

    PubMed

    Espinosa Neira, Roberto; Salazar, Eduardo Perez

    2012-12-01

    Basement membrane (BM) is a complex network of interacting proteins, including type IV collagen (Col IV) that acts as a scaffold that stabilizes the physical structures of tissues and regulates cellular processes. In the mammary gland, BM is a continuous deposit that separates epithelial cells from stroma, and its degradation is related with an increased potential for invasion and metastasis. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells are transdifferentiated to one mesenchymal state, and is a normal process during embryonic development, tissue remodeling and wound healing, as well as it has been implicated during cancer progression. In breast cancer cells, native Col IV induces migration and gelatinases secretion. However, the role of native Col IV on the EMT process in human mammary epithelial cells remains to be investigated. In the present study, we demonstrate that native Col IV induces down-regulation of E-cadherin expression, accompanied with an increase of Snail1, Snail2 and Sip1 transcripts. Native Col IV also induces an increase in N-cadherin and vimentin expression, an increase of MMP-2 secretion, the activation of FAK and NFκB, cell migration and invasion in MCF10A cells. In summary, these findings demonstrate, for the first time, that native Col IV induces an EMT-like process in MCF10A human mammary non-tumorigenic epithelial cells. PMID:22981734

  6. Proteomic profiling of acrolein adducts in human lung epithelial cells

    PubMed Central

    Spiess, Page C.; Deng, Bin; Hondal, Robert J.; Matthews, Dwight E.; van der Vliet, Albert

    2011-01-01

    Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology. PMID:21704744

  7. Plasticity of Airway Epithelial Cell Transcriptome in Response to Flagellin

    PubMed Central

    Clark, Joan G.; Kim, Kyoung-Hee; Basom, Ryan S.; Gharib, Sina A.

    2015-01-01

    Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture versus ALI culture using whole-genome microarrays and RNA sequencing. We exposed monolayer and ALI AEC cultures to flagellin in vitro and analyzed the transcriptional response by microarray and RNA-sequencing. ELISA and RT-PCR were used to validate changes in select candidates. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment of inflammatory and immune pathways. We conclude that in vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium. PMID:25668187

  8. Sprayed cultured mucosal epithelial cell for deep dermal burns.

    PubMed

    Ueda, Minoru

    2010-11-01

    Mucosal epithelial cells have various advantages compared with epidermal cells, such as their high proliferation ability and long biologic activity. The objective of this study was to assess the clinical results after sprayed application of cultured mucosal epithelial autograft (CMEA) suspensions onto deep dermal burn wounds. Ten patients with deep dermal burns were included in a prospective study. The average total-body-surface-area burn was 17.7% (8%-45%). The average Abbreviated Burn Severity Index was 6.3 points (4-9 points). The application of sprayed CMEA suspension was performed onto an average body surface area of 2.05% (0.5%-5%; median, 2%). Eight patients were recruited for clinical follow-up after an average of 10 months (3-18 months). The average Vancouver Scar Scale score at follow-up was 1.5 points (range, 0-5 points). The average period of epithelialization in wound surface was 12.5 days. Our data show that enzymatic and careful surgical debridement and consecutive application of CMEA suspensions using a spray technique result in excellent cosmetic outcomes compared with any other methods. PMID:21119409

  9. Intracellular autofluorescence: a biomarker for epithelial cancer stem cells.

    PubMed

    Miranda-Lorenzo, Irene; Dorado, Jorge; Lonardo, Enza; Alcala, Sonia; Serrano, Alicia G; Clausell-Tormos, Jenifer; Cioffi, Michele; Megias, Diego; Zagorac, Sladjana; Balic, Anamaria; Hidalgo, Manuel; Erkan, Mert; Kleeff, Joerg; Scarpa, Aldo; Sainz, Bruno; Heeschen, Christopher

    2014-11-01

    Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells. PMID:25262208

  10. Comparison of upstream regulators in human ex vivo cultured cornea limbal epithelial stem cells and differentiated corneal epithelial cells

    PubMed Central

    2013-01-01

    Background The surface of the human eye is covered by corneal epithelial cells (CECs) which regenerate from a small population of limbal epithelial stem cells (LESCs). Cell therapy with LESCs is a non-penetrating treatment for preventing blindness due to LESC deficiency or dysfunction. Our aim was to identify new putative molecular markers and upstream regulators in the LESCs and associated molecular pathways. Results Genome-wide microarray transcriptional profiling was used to compare LESCs to differentiated human CECs. Ingenuity-based pathway analysis was applied to identify upstream regulators and pathways specific to LESCs. ELISA and flow cytometry were used to measure secreted and surface expressed proteins, respectively. More than 2 fold increase and decrease in expression could be found in 1830 genes between the two cell types. A number of molecules functioning in cellular movement (381), proliferation (567), development (552), death and survival (520), and cell-to-cell signaling (290) were detected having top biological functions in LESCs and several of these were confirmed by flow cytometric surface protein analysis. Custom-selected gene groups related to stemness, differentiation, cell adhesion, cytokines and growth factors as well as angiogenesis could be analyzed. The results show that LESCs play a key role not only in epithelial differentiation and tissue repair, but also in controlling angiogenesis and extracellular matrix integrity. Some pro-inflammatory cytokines were found to be important in stemness-, differentiation- and angiogenesis-related biological functions: IL-6 and IL-8 participated in most of these biological pathways as validated by their secretion from LESC cultures. Conclusions The gene and molecular pathways may provide a more specific understanding of the signaling molecules associated with LESCs, therefore, help better identify and use these cells in the treatment of ocular surface diseases. PMID:24344983

  11. Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

    PubMed Central

    Vassilieva, Elena V.; Ivanov, Andrei I.; Nusrat, Asma

    2010-01-01

    Flotillins and caveolins represent two types of resident proteins associated with lipid rafts in mammalian cells, however, their possible cross-talk in regulating lipid raft functions remains poorly understood. In this report, we observed that siRNA-mediated down-regulation of flotillin-1 expression which disrupted lipid raft-mediated endocytosis of BODIPY FL C5-lactosylceramide also substantially decreased caveolin-1 level in SK-CO15 human intestinal epithelial cells. The decrease in caveolin-1 expression appeared to be specific for flotillin-1 knock-down and was not observed after down-regulation of flotillin-2. The decrease in caveolin-1 level in flotillin-1-depleted cells was not due to suppression of its mRNA synthesis and was not mimicked by cholesterol depletion of SK-CO15 cells. Furthermore, flotillin-1 dependent down-regulation of caveolin-1 was reversed after cell exposure to lysosomal inhibitor, chloroquine but not proteosomal inhibitor, MG262. Our data suggest that flotillin-1 regulates caveolin-1 level by preventing its lysosomal degradation in intestinal epithelial cells. PMID:19121286

  12. Technical note: isolation and characterization of sheep ruminal epithelial cells.

    PubMed

    Baldwin, R L; Jesse, B W

    1991-09-01

    A system for the isolation and characterization of sheep ruminal epithelial cells has been developed. Ruminal papillae from the ventral cranial sac of 12- to 24-wk-old Dorset ram lambs were subjected to serial tryptic digestions. Initial digestions (two 15-min periods) in the series were used to remove keratinized cells of the stratum corneum, which were able to produce only small amounts of beta-hydroxybutyrate from butyrate (5.37 +/- .72 nmol/120 min/per milligram of dry cell weight). Later digest fractions, containing primarily cells from the stratum basale, exhibited high viabilities (75 to 95%) and proved capable of converting butyrate to beta-hydroxybutyrate at relatively high rates (33.6 +/- 6.7 nmol/120 min per milligram of dry cell weight). Neither acetate nor propionate underwent significant conversion to beta-hydroxybutyrate. However, addition of acetate inhibited (77.4% of control) and addition of propionate stimulated (200% of control) beta-hydroxybutyrate production. Acetate addition reduced the propionate-induced stimulation of beta-hydroxybutyrate production from butyrate (136% of control). These results are similar to those obtained from in vitro incubations of ruminal papillae and suggest that an isolated cell system may prove useful in the further study of ruminal epithelial metabolism. PMID:1938645

  13. ATP7B detoxifies silver in ciliated airway epithelial cells.

    PubMed

    Ibricevic, Aida; Brody, Steven L; Youngs, Wiley J; Cannon, Carolyn L

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B(-/-) mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag+/Cu+ transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung. PMID:20005242

  14. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  15. 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 showed signs of damage by viruses and virus titers (see figure) that indicated large increases in the populations of viruses during the days following inoculation.

  16. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    EPA Science Inventory

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

  17. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  18. Hypoxia?inducible adrenomedullin ameliorates the epithelial-to-mesenchymal transition in human proximal tubular epithelial cells.

    PubMed

    Zhu, Tiechui; Yang, Jun; Liu, Xiangdong; Zhang, Lianyun; Zhang, Jie; Wang, Yongtao; Ma, Haijun; Ren, Zhenhui

    2015-05-01

    Renal tubular epithelial cells can enter the epithelial?to?mesenchymal transition (EMT) in response to chronic hypoxia. EMT is a process which involves the phenotypic conversion of epithelial cells, that is believed to have an important role in renal fibrosis. However, the underlying mechanisms of the involvement of EMT in renal fibrosis remain to be elucidated. Adrenomedullin (AMD) has been implicated in renal fibrosis and is induced by hypoxia. The aims of the present study were to determine whether ADM signaling was active in human proximal tubular epithelial cells cultured under hypoxic conditions, and to observe the activity of ADM during EMT. The expression levels of ADM were significantly increased, in a time?dependent manner, in HK?2 and HKC human proximal tubular epithelial cells, cultured under hypoxic conditions. Overexpression of exogenous ADM was accompanied by increased expression levels of the epithelial markers E?cadherin and tight junction protein?1, and decreased expression levels of the mesenchymal markers vimentin and ??smooth muscle actin, during hypoxia. Knock?down of ADM expression by small hairpin RNA, or co?administration of an ADM peptide inhibitor, in HK?2 cells significantly exacerbated hypoxia?induced EMT, as compared to the lack of effect observed in the untransfected controls. ADM was shown to suppress EMT by inhibiting the activation of extracellular signal?regulated kinase (ERK), and this effect was prevented by the ERK activator apigenin. The results of the present study suggest that ADM has an important role in promoting EMT in hypoxic human proximal tubular epithelial cells. ADM may therefore represent a novel therapeutic target in the treatment of injured kidneys. PMID:25586428

  19. Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells.

    PubMed

    Hyun, Dong Won; Kim, Yun Hee; Koh, Ah Young; Lee, Hyun Ju; Wee, Won Ryang; Jeon, Saewha; Kim, Mee Kum

    2014-11-19

    The purpose of this study was to report the characteristics of biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal-deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony-forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki-67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial-free sheets. Biomaterial-free sheets which had been detached with 1% dispase or biomaterial-free sheets generated by fibrin support were transplanted to 12 limbal-deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial-free sheets were compared with those of sheets formed by fibrin-coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial-free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial-free sheets were comparable to those of sheets cultured by fibrin support, in limbal-deficient models. Both p63 and Ki-67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal-deficient models. Our results suggest that biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal-deficient diseases. Copyright 2014 John Wiley & Sons, Ltd. PMID:25407749

  20. How Bacteria-Induced Apoptosis of Intestinal Epithelial Cells Contributes to Mucosal Inflammation

    PubMed Central

    Hausmann, Martin

    2010-01-01

    The life cycle of an intestinal epithelial cell is terminated by apoptosis and/or cell shedding. Apoptotic deletion of epithelial cells from the intact intestinal mucosa is not accompanied by detectable inflammatory response or loss of barrier function. But increased permeability of the epithelial barrier and increased apoptotic rates of epithelial cells have been reported for patients suffering from inflammatory bowel disease. Microbiota can both induce or inhibit apoptosis of intestinal epithelial cells thus contribute to mucosal inflammation or support epithelial integrity respectively. Bacteria-mediated cytokine secretion and altered cell signalling are central to epithelial injury. Tumor necrosis factor (TNF) secreted after exposure to invasive bacteria induces both apoptosis and cell shedding. TNF is the major target gene of the transcription factor nuclear factor-kappa B with both pro- and anti-apoptotic effects. Autophagy promotes both cell survival and autophagic cell death. If autophagy is directed against microbes it is termed xenophagy. Inhibition of xenophagy has been shown to decrease cell survival. Endoplasmic reticulum (ER) stress causes misfolded proteins to accumulate in the ER lumen. It was suggested that ER stress and autophagy may interact within intestinal epithelial cells. Apoptosis in response to infection may be well proposed by the host to delete infected epithelial cells or could be a strategy of microbial pathogens to escape from exhausted cells to invade deeper mucosal layers for a prolonged bacterial colonization. PMID:21188215

  1. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  2. IL-4 Attenuates Pulmonary Epithelial Cell-Mediated Suppression of T Cell Priming

    PubMed Central

    Albrecht, Melanie; Arnhold, Markus; Lingner, Sandra; Mahapatra, Subhashree; Bruder, Dunja; Hansen, Gesine; Dittrich, Anna-Maria

    2012-01-01

    We have previously shown that Th2-polarized airway inflammation facilitates sensitization towards new, protein antigens. In this context, we could demonstrate that IL-4 needs to act on cells of the hematopoetic and the structural compartment in order to facilitate sensitization towards new antigens. We thus aimed to elucidate possible mechanisms of action of IL-4 on structural cells choosing to analyze pulmonary epithelial cells as an important part of the lung's structural system. We used a co-culture system of DC- or APC-dependent in vitro priming of T cells, co-cultivated on a layer of cells of a murine pulmonary epithelial cell line (LA-4) pretreated with or without IL-4. Effects on T cell priming were analyzed via CFSE-dilution and flow cytometric assessment of activation status. Pulmonary epithelial cells suppressed T cell proliferation in vitro but this effect was attenuated by pre-treatment of the epithelial cells with IL-4. Transwell experiments suggest that epithelial-mediated suppression of T cell activation is mostly cell-contact dependent and leads to attenuation in an early naive T cell phenotype. Secretion of soluble factors like TARC, TSLP, GM-CSF and CCL20 by epithelial cells did not change after IL-4 treatment. However, analysis of co-stimulatory expression on pulmonary epithelial cells revealed that pre-treatment of epithelial cells with IL-4 changed expression GITR-L, suggesting a possible mechanism for the effects observed. Our studies provide new insight into the role of IL-4 during the early phases of pulmonary sensitization: The inhibitory activity of pulmonary epithelial cells in homeostasis is reversed in the presence of IL-4, which is secreted in the context of Th2-dominated allergic airway inflammation. This mechanism might serve to explain facilitated sensitization in the clinical context of polysensitization where due to a pre-existing sensitization increased levels of IL-4 in the airways might facilitate T cell priming towards new antigens. PMID:23029313

  3. Isoprenaline induces epithelial-mesenchymal transition in gastric cancer cells.

    PubMed

    Lu, Yan-Jie; Geng, Zhi-Jun; Sun, Xiao-Yan; Li, Yu-Hong; Fu, Xiao-Bing; Zhao, Xiang-Yang; Wei, Bo

    2015-10-01

    The emerging role of stress-related signaling in regulating cancer development and progression has been recognized. However, whether stress serves as a mechanism to promote gastric cancer metastasis is not clear. Here, we show that the β2-AR agonist, isoprenaline, upregulates expression levels of CD44 and CD44v8-10 in gastric cancer cells. CD44, a cancer stem cell-related marker, is expressed at high levels in gastric cancer tissues, which strongly correlates with the occurrence of epithelial-mesenchymal transition (EMT)-associated phenotypes both in vivo and in vitro. Combined with experimental observations in two human gastric cancer cell lines, we found that β2-AR signaling can initiate EMT. It led to an increased expression of mesenchymal markers, such as α-SMA, vimentin, and snail at mRNA and protein levels, and conversely a decrease in epithelial markers, E-cadherin and β-catenin. Isoprenaline stimulation of β2-AR receptors activates the downstream target STAT3, which functions as a positive regulator and mediated the phenotypic switch toward a mesenchymal cell type in gastric cancer cells. Our data provide a mechanistic understanding of the complex signaling cascades involving stress-related hormones and their effects on EMT. In light of our observations, pharmacological interventions targeting β2-AR-STAT3 signaling can potentially be used to ameliorate stress-associated influences on gastric cancer development and progression. PMID:26253173

  4. Biomarkers of Epithelial-Mesenchymal Transition in Squamous Cell Carcinoma

    PubMed Central

    Scanlon, C.S.; Van Tubergen, E.A.; Inglehart, R.C.; DSilva, N.J.

    2013-01-01

    An understanding of the process by which tumor cells destroy the basement membrane of the surface epithelium, invade, and metastasize is essential to the development of novel treatment of head and neck squamous cell carcinoma (HNSCC). In recent years, there has been increased interest in the role of epithelial-mesenchymal transition (EMT) in invasion. EMT is a process that describes the development of motile, mesenchymal-like cells from non-motile parent epithelial cells. There are 3 known types of EMT that mediate development, wound healing, and carcinogenesis. This review summarizes studies of known EMT biomarkers in the context of HNSCC progression. The biomarkers discussed come from a wide range of proteins, including cell-surface proteins (E-cadherin, N-cadherin, and Integrins), cytoskeletal proteins (?-Smooth Muscle Actin, Vimentin, and ?-catenin), extracellular matrix proteins (Collagens, Fibronectin, and Laminin), and transcription factors (SNAIL1, SNAIL2, TWIST, and LEF-1). Overall, the findings of these studies suggest that EMT mediates HNSCC progression. The mechanistic role of the EMT markers that have been associated with HNSCC should be more clearly defined if new anti-HNSCC therapies to block EMT progression are to be developed. PMID:23128109

  5. Nectin 4 is the epithelial cell receptor for measles virus.

    PubMed

    Noyce, Ryan S; Richardson, Christopher D

    2012-09-01

    Measles virus (MV) causes acute respiratory disease, infects lymphocytes and multiple organs, and produces immune suppression leading to secondary infections. In rare instances it can also cause persistent infections in the brain and central nervous system. Vaccine and laboratory-adapted strains of MV use CD46 as a receptor, whereas wild-type strains of MV (wtMV) cannot. Both vaccine and wtMV strains infect lymphocytes, monocytes, and dendritic cells (DCs) using the signaling lymphocyte activation molecule (CD150/SLAM). In addition, MV can infect the airway epithelial cells of the host. Nectin 4 (PVRL4) was recently identified as the epithelial cell receptor for MV. Coupled with recent observations made in MV-infected macaques, this discovery has led to a new paradigm for how the virus accesses the respiratory tract and exits the host. Nectin 4 is also a tumor cell marker which is highly expressed on the apical surface of many adenocarcinoma cell lines, making it a potential target for MV oncolytic therapy. PMID:22721863

  6. Temporal association of serum progesterone concentrations and vaginal cytology in walruses (Odobenus rosmarus).

    PubMed

    Kinoshita, K; Kiwata, M; Kuwano, R; Sato, N; Tanaka, T; Nagata, M; Taira, H; Kusunoki, H

    2012-03-15

    Concentrations of serum estradiol-17? and progesterone were monitored in six female walruses using an enzyme immunoassay. Progesterone concentrations increased from March to May in females aged 6 y or older, and subsequently declined (October). No significant elevation of estradiol-17? concentration was detected before an elevation of progesterone concentration. Vaginal smears from four females were examined with Papanicolaou staining. In all females, most epithelial cells were basophilic intermediate-superficial cells; no color change from basophilic to eosinophilic of the cells was detected. Meanwhile, the percentage of anucleate cells in vaginal smears reached its highest value before the elevation of progesterone concentration, followed by an increase in the percentage of leukocytes. We inferred that the change in populations of anucleate cells and leukocytes in vaginal smears reflected ovarian status and CL formation in female walruses. PMID:22153266

  7. Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool.

    PubMed

    Mesa, Kailin R; Rompolas, Panteleimon; Zito, Giovanni; Myung, Peggy; Sun, Thomas Y; Brown, Samara; Gonzalez, David G; Blagoev, Krastan B; Haberman, Ann M; Greco, Valentina

    2015-06-01

    Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression). In contrast to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration. Here we show by intravital microscopy in live mice that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbours. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through transforming growth factor (TGF)-? activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool, as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviours and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis. PMID:25849774

  8. In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

    PubMed

    Isaacson, R E; Fusco, P C; Brinton, C C; Moon, H W

    1978-08-01

    Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili. PMID:357285

  9. Estrogen Vaginal

    MedlinePLUS

    ... estradiol vaginal ring is also used to treat hot flushes ('hot flashes'; sudden strong feelings of heat and sweating) ... mild soap and warm water. Do not use hot water or boil the applicator. Ask your pharmacist ...

  10. Vaginal Bleeding

    MedlinePLUS

    ... or period, is a woman's monthly bleeding. Abnormal vaginal bleeding is different from normal menstrual periods. It ... therapy Cancer of the cervix, ovaries, uterus or vagina Thyroid problems Bleeding during pregnancy can have several ...

  11. Vaginal Pessary

    MedlinePLUS

    ... your vagina). A pessary can also help many women who have stress urinary incontinence (the leaking of urine when you cough, strain or exercise). Pregnant women who have incontinence can also use a vaginal ...

  12. Vaginal Discharge

    MedlinePLUS

    ... in vaginal discharge? Changes can occur if the normal balance of healthy bacteria (germs) in your vagina is ... douches may irritate your vagina and change the normal balance of germs in your vagina. Douching can also ...

  13. Vaginal Cancer

    MedlinePLUS

    ... medical, surgical, radiation, gynecologic, and pediatric oncologists, oncology nurses, physician assistants, social workers, and patient advocates. Cancer.Net Guide Vaginal Cancer ... with Side Effects After Treatment Questions to Ask ...

  14. Inflammatory response modulation of airway epithelial cells exposed to formaldehyde.

    PubMed

    Persoz, Charles; Achard, Sophie; Momas, Isabelle; Seta, Nathalie

    2012-06-01

    The two main difficulties when assessing the role and action mechanism of environmental pollutant exposure on the respiratory tract using in vitro methodology are firstly to create exposure conditions that closely mimic the human situation, and secondly to choose an experimental model that accurately represents lung compartment complexity, with different types of cell interaction. The aim of this study was to resolve these two challenges. The first of our difficulties was to find the closest experimental conditions to mimic respiratory environmental pollutant exposure. We compared the effects of formaldehyde (FA) on two cellular models, alveolar and bronchial cell lines, respectively A549 and BEAS-2B. The cells were exposed for 30 min to an environmental dose of gaseous FA (50 μg/m³) at the air-liquid interface. In order to mimic macrophage-epithelial cell cooperation, sensitizations (with TNFα or with conditioned medium from macrophages--CM) prior to gas exposure were applied. After toxicity evaluation, local inflammation was assessed by IL-8 and MCP-1 production 24h after exposure. In our experimental conditions FA had no effects on alveolar and bronchial epithelial cells without any sensitization. FA exposure after TNFα sensitization alone induced a moderate increase of IL-8 by A549 cells. After sensitization with CM, FA exposure induced a strong increase of IL-8 production by A549 cells in comparison to air, whereas a decrease of MCP-1 production was observed on BEAS-2B cells. It appears that the response of alveolar and bronchial epithelial cells to FA was moderate and that complex sensitization refines the inflammatory response to environmental stresses. When sensitized with CM, these cell lines responded differently to FA exposure. Finally by interacting with the respiratory epithelium, FA could exacerbate the inflammation of airways that occurs in severe asthma, and even synergize the effects of other air pollutants such as allergens. Evaluation of nasal cell inflammatory response could shed further light on the effects of FA on respiratory epithelium. PMID:22484645

  15. Hysterectomy - vaginal - discharge

    MedlinePLUS

    Vaginal hysterectomy - discharge; Laparoscopically assisted vaginal hysterectomy - discharge; LAVH - discharge ... you were in the hospital, you had a vaginal hysterectomy. Your surgeon made a cut in your ...

  16. Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

    NASA Astrophysics Data System (ADS)

    Chen, Kun; Qin, Yejun; Zheng, Feng; Sun, Menghong; Shi, Daren

    2006-07-01

    A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis.

  17. Vaginal reconstruction

    SciTech Connect

    Lesavoy, M.A.

    1985-05-01

    Vaginal reconstruction can be an uncomplicated and straightforward procedure when attention to detail is maintained. The Abbe-McIndoe procedure of lining the neovaginal canal with split-thickness skin grafts has become standard. The use of the inflatable Heyer-Schulte vaginal stent provides comfort to the patient and ease to the surgeon in maintaining approximation of the skin graft. For large vaginal and perineal defects, myocutaneous flaps such as the gracilis island have been extremely useful for correction of radiation-damaged tissue of the perineum or for the reconstruction of large ablative defects. Minimal morbidity and scarring ensue because the donor site can be closed primarily. With all vaginal reconstruction, a compliant patient is a necessity. The patient must wear a vaginal obturator for a minimum of 3 to 6 months postoperatively and is encouraged to use intercourse as an excellent obturator. In general, vaginal reconstruction can be an extremely gratifying procedure for both the functional and emotional well-being of patients.

  18. EGF stimulates Mg(2+) influx in mammary epithelial cells.

    PubMed

    Trapani, Valentina; Arduini, Daniela; Luongo, Francesca; Wolf, Federica I

    2014-10-30

    Magnesium is well established as a fundamental factor that regulates cell proliferation. However, the molecular mechanisms linking mitogenic signals, extracellular magnesium availability and intracellular effectors are still largely unknown. In the present study we sought to determine whether EGF regulates magnesium homeostasis in normal HC11 mammary epithelial cells. To this end, we measured Mg(2+) and Ca(2+) fluxes by confocal imaging in live cells loaded with specific fluorescent ion indicators (Mag-Fluo-4 and Fluo-4, respectively). EGF stimulation induces a rapid and sustained increase in intracellular Mg(2+), concomitantly with a rise in intracellular calcium. The increase in intracellular Mg(2+) derives from an influx from the extracellular compartment, and does not depend on Ca(2+). On the contrary, the increase in intracellular Ca(2+) derives from intracellular stores, and is impaired in the absence of extracellular magnesium. Inhibition of the EGF receptor tyrosine kinase by Tyrphostin AG1478 markedly inhibits EGF-induced Mg(2+) and Ca(2+) signals. These findings demonstrate that not only does Mg(2+) influx represent an important step in the physiological response of epithelial cells to EGF, but unexpectedly the EGF-induced Mg(2+) influx is essential for the Ca(2+) signal to occur. PMID:25450695

  19. OCRL1 modulates cilia length in renal epithelial cells.

    PubMed

    Rbaibi, Youssef; Cui, Shanshan; Mo, Di; Carattino, Marcelo; Rohatgi, Rajeev; Satlin, Lisa M; Szalinski, Christina M; Swanhart, Lisa M; Flsch, Heike; Hukriede, Neil A; Weisz, Ora A

    2012-09-01

    Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome. PMID:22680056

  20. OCRL1 Modulates Cilia Length in Renal Epithelial Cells

    PubMed Central

    Rbaibi, Youssef; Cui, Shanshan; Mo, Di; Carattino, Marcelo; Rohatgi, Rajeev; Satlin, Lisa M.; Szalinski, Christina M.; Swanhart, Lisa M.; Fölsch, Heike; Hukriede, Neil A.; Weisz, Ora A.

    2013-01-01

    Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome. PMID:22680056

  1. Multicolor Cell Barcoding Technology for Long-Term Surveillance of Epithelial Regeneration in Zebrafish.

    PubMed

    Chen, Chen-Hui; Puliafito, Alberto; Cox, Ben D; Primo, Luca; Fang, Yi; Di Talia, Stefano; Poss, Kenneth D

    2016-03-21

    Current fate mapping and imaging platforms are limited in their ability to capture dynamic behaviors of epithelial cells. To deconstruct regenerating adult epithelial tissue at single-cell resolution, we created a multicolor system, skinbow, that barcodes the superficial epithelial cell (SEC) population of zebrafish skin with dozens of distinguishable tags. With image analysis to directly segment and simultaneously track hundreds of SECs in vivo over entire surface lifetimes, we readily quantified the orchestration of cell emergence, growth, repositioning, and loss under homeostatic conditions and after exfoliation or appendage amputation. We employed skinbow-based imaging in conjunction with a live reporter of epithelial stem cell cycle activity and as an instrument to evaluate the effects of reactive oxygen species on SEC behavior during epithelial regeneration. Our findings introduce a platform for large-scale, quantitative in vivo imaging of regenerating skin and reveal unanticipated collective dynamism in epithelial cell size, mobility, and interactions. PMID:27003938

  2. Force dependence of phagosome trafficking in retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Daniel, Rebekah; Koll, Andrew T.; Altman, David

    2014-09-01

    Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

  3. 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

  4. Plasticity of epithelial stem cells in tissue regeneration

    PubMed Central

    Blanpain, Cédric; Fuchs, Elaine

    2015-01-01

    Tissues rely upon stem cells for homeostasis and repair. Recent studies show that the fate and multilineage potential of epithelial stem cells can change depending on whether a stem cell exists within its resident niche and responds to normal tissue homeostasis, whether it is mobilized to repair a wound, or whether it is taken from its niche and challenged to de novo tissue morphogenesis after transplantation. In this Review, we discuss how different populations of naturally lineage-restricted stem cells and committed progenitors can display remarkable plasticity and reversibility and reacquire long-term self-renewing capacities and multilineage differentiation potential during physiological and regenerative conditions. We also discuss the implications of cellular plasticity for regenerative medicine and for cancer. PMID:24926024

  5. Limbal stem cells: Central concepts of corneal epithelial homeostasis.

    PubMed

    Yoon, Jinny J; Ismail, Salim; Sherwin, Trevor

    2014-09-26

    A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface. PMID:25258661

  6. Generating retinal neurons by reprogramming retinal pigment epithelial cells

    PubMed Central

    Wang, Shu-Zhen; Ma, Wenxin; Yan, Run-Tao; Mao, Weiming

    2010-01-01

    Importance of the field Retinal degenerations cause blindness. One potential therapy is cell replacement. Because the human retina lacks regeneration capacity, much attention has been directed towards searching for cells that can differentiate into retinal neurons. Areas covered in this review We discuss the possibility of using transcription factor genes to channel retinal pigment epithelial (RPE) cells capabilities of proliferation and plasticity towards the production of retinal neurons. What the reader will gain Experiments with chick embryos show that RPE cells in the eye, in explant, or in a dissociated cell culture can give rise to cells resembling retinal neurons when reprogrammed with regulatory genes involved in retinal neurogenesis. Depending on the regulatory gene used, reprogramming generates cells exhibiting traits of photoreceptor cells, amacrine cells and/or young ganglion neurons. Take home message Gene-directed reprogramming of chick RPE can efficiently generate cells that exhibit traits of retinal neurons. Remaining to be addressed is the question of whether the results from chicks apply to mammals. Since the RPE is located adjacent to the neural retina, RPE reprogramming, if successful in mammals, may offer an approach to repopulate the neural retina without involving cell transplantation. PMID:20528097

  7. Limbal stem cells: Central concepts of corneal epithelial homeostasis

    PubMed Central

    Yoon, Jinny J; Ismail, Salim; Sherwin, Trevor

    2014-01-01

    A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface PMID:25258661

  8. ZAS3 Accentuates Transforming Growth Factor ? Signaling in Epithelial Cells

    PubMed Central

    Yakovich, Adam J.; Jiang, Bo; Allen, Carl E; Du, Jianguo; Wu, Lai-Chu; Barnard, John A.

    2010-01-01

    In mammals, the ZAS family of transcription factors activates or represses transcription depending on the cellular context. In the current study, we explored the interaction between ZAS3 and TGF?1 signaling in epithelial cells using HEK293 cells and the intestinal epithelial cell line, RIE-1. Endogenous ZAS3 expression was detected in each cell line and the small intestine of mice. Additionally, endogenous ZAS3 expression was increased in both whole cell and nuclear lysates by TGF?1 and in vivo in TGF?-overexpressing mice, indicating a potential interaction between ZAS3 and TGF?. ZAS3 transfection enhanced TGF?1 activation of a luciferase reporter in both HEK293 and RIE-1 cells. Analysis of truncated ZAS3 constructs revealed a 155 amino acid, N-terminal sequence between amino acids 106 and 261 that was required for enhancement of TGF?1-mediated transcription. Coimmunoprecipitation experiments with nuclear extracts from TGF?1-stimulated HEK293 cells revealed an association between ZAS3 and the Smad complex. Additionally, transfected ZAS3 decreased the association between the Smad complex and the TGF? transcriptional repressors Ski and SnoN, indicating a possible mechanism for the enhancement of transcription by exogenous ZAS3. These observations were confirmed by site-directed mutagenesis of ZAS domains homologous with Smad-interacting domains in Ski and SnoN. Finally, ZAS3 transfection enhanced the TGF?1-mediated induction of ?-smooth muscle actin in HEK293 cells, indicating that ZAS3 plays a functional role in TGF? signaling. In conclusion, we have identified an interaction between ZAS3 and Smad proteins that enhances TGF? signaling. Since TGF? signaling is primarily known as a negatively regulated pathway, the enhancement of signaling by ZAS3 has novel implications for understanding TGF? biology. PMID:20732416

  9. Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells.

    PubMed

    Roberts, Joan E; Wielgus, Albert R; Boyes, William K; Andley, Usha; Chignell, Colin F

    2008-04-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C60(OH)22-26] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C60(OH)22-26 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 microM. Exposure to either UVA or visible light in the presence of >5 microM fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 microM lutein, 1 mM N-acetyl cysteine, or 1 mM l-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA>visible light>dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein alpha-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C60(OH)22-26 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo. PMID:18234258

  10. Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

    SciTech Connect

    Roberts, Joan E. Wielgus, Albert R. Boyes, William K. Andley, Usha Chignell, Colin F.

    2008-04-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C{sub 60}(OH){sub 22-26}] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C{sub 60}(OH){sub 22-26} in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 {mu}M. Exposure to either UVA or visible light in the presence of > 5 {mu}M fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 {mu}M lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein {alpha}-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C{sub 60}(OH){sub 22-26} is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo.

  11. Phototoxicity and Cytotoxicity of Fullerol in Human Lens Epithelial Cells

    PubMed Central

    Roberts, Joan E.; Wielgus, Albert R.; Boyes, William K.; Andley, Usha; Chignell, Colin F.

    2008-01-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C60(OH)2226] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerols potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C60(OH)2226 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 M. Exposure to either UVA or visible light in the presence of >5 M fullerol induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 M lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein ?-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water soluble nano-C60(OH)2226 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo. PMID:18234258

  12. In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

    PubMed Central

    Hu, Han; Wang, Jiaqi; Bu, Dengpan; Wei, Hongyang; Zhou, Linyun; Li, Fadi; Loor, Juan J.

    2009-01-01

    Background The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. Methodology Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. Principal Findings The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n?=?60. Furthermore, they were capable of synthesizing ?-casein (CSN2), acetyl-CoA carboxylase-? (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. Conclusions The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs). PMID:19888476

  13. Cell crawling mediates collective cell migration to close undamaged epithelial gaps.

    PubMed

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C; Sheetz, Michael P; Trepat, Xavier; Ladoux, Benot

    2012-07-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 ?m was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  14. Epithelial odontogenic ghost cell tumour of the mandibular gingiva.

    PubMed

    Lombardi, T; Kffer, R; Di Felice, R; Samson, J

    1999-07-01

    The epithelial odontogenic ghost cell tumour (EOGCT) is considered as a solid 'neoplastic' variant of the calcifying odontogenic cyst and is an uncommon lesion for which various names have been proposed over the years. We describe here an extraosseous case occurring on the edentulous mandibular gingiva in the right bicuspid area of a 70-year-old woman. The lesion was a painless nodule that appeared clinically as a hyperplastic mass, which was considered to be of reactive nature. Radiographic examination showed a localised resorption of the underlying mandibular bone. The tumour was excised; there was no recurrence at a 2-year follow-up examination. PMID:10645413

  15. Comparative proteomics reveals human pluripotent stem cell-derived limbal epithelial stem cells are similar to native ocular surface epithelial cells.

    PubMed

    Mikhailova, Alexandra; Jylh, Antti; Rieck, Jochen; Nttinen, Janika; Ilmarinen, Tanja; Verb, Zoltn; Aapola, Ulla; Beuerman, Roger; Petrovski, Goran; Uusitalo, Hannu; Skottman, Heli

    2015-01-01

    Limbal epithelial stem cells (LESCs) are tissue-specific stem cells responsible for renewing the corneal epithelium. Acute trauma or chronic disease affecting LESCs may disrupt corneal epithelial renewal, causing vision threatening and painful ocular surface disorders, collectively referred to as LESC deficiency (LESCD). These disorders cannot be treated with traditional corneal transplantation and therefore alternative cell sources for successful cell-based therapy are needed. LESCs derived from human pluripotent stem cells (hPSCs) are a prospective source for ocular surface reconstruction, yet critical evaluation of these cells is crucial before considering clinical applications. In order to quantitatively evaluate hPSC-derived LESCs, we compared protein expression in native human corneal cells to that in hPSC-derived LESCs using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We identified 860 unique proteins present in all samples, including proteins involved in cell cycling, proliferation, differentiation and apoptosis, various LESC niche components, and limbal and corneal epithelial markers. Protein expression profiles were nearly identical in LESCs derived from two different hPSC lines, indicating that the differentiation protocol is reproducible, yielding homogeneous cell populations. Their protein expression profile suggests that hPSC-derived LESCs are similar to the human ocular surface epithelial cells, and possess LESC-like characteristics. PMID:26423138

  16. Comparative proteomics reveals human pluripotent stem cell-derived limbal epithelial stem cells are similar to native ocular surface epithelial cells

    PubMed Central

    Mikhailova, Alexandra; Jylhä, Antti; Rieck, Jochen; Nättinen, Janika; Ilmarinen, Tanja; Veréb, Zoltán; Aapola, Ulla; Beuerman, Roger; Petrovski, Goran; Uusitalo, Hannu; Skottman, Heli

    2015-01-01

    Limbal epithelial stem cells (LESCs) are tissue-specific stem cells responsible for renewing the corneal epithelium. Acute trauma or chronic disease affecting LESCs may disrupt corneal epithelial renewal, causing vision threatening and painful ocular surface disorders, collectively referred to as LESC deficiency (LESCD). These disorders cannot be treated with traditional corneal transplantation and therefore alternative cell sources for successful cell-based therapy are needed. LESCs derived from human pluripotent stem cells (hPSCs) are a prospective source for ocular surface reconstruction, yet critical evaluation of these cells is crucial before considering clinical applications. In order to quantitatively evaluate hPSC-derived LESCs, we compared protein expression in native human corneal cells to that in hPSC-derived LESCs using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We identified 860 unique proteins present in all samples, including proteins involved in cell cycling, proliferation, differentiation and apoptosis, various LESC niche components, and limbal and corneal epithelial markers. Protein expression profiles were nearly identical in LESCs derived from two different hPSC lines, indicating that the differentiation protocol is reproducible, yielding homogeneous cell populations. Their protein expression profile suggests that hPSC-derived LESCs are similar to the human ocular surface epithelial cells, and possess LESC-like characteristics. PMID:26423138

  17. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells.

    PubMed

    Huang, Minghai; Wang, Bowen; Wan, Pengxia; Liang, Xuanwei; Wang, Xiaoran; Liu, Ying; Zhou, Qiang; Wang, Zhichong

    2015-02-01

    Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future. PMID:25398719

  18. Barrier Epithelial Cells and the Control of Type 2 Immunity.

    PubMed

    Hammad, Hamida; Lambrecht, Bart N

    2015-07-21

    Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4(+) T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-mediated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mechanisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads to protection or disease. PMID:26200011

  19. Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures

    PubMed Central

    Cui, Xiaohong; Lee, Ronzo; Adler, Stuart P.; McVoy, Michael A.

    2013-01-01

    Anti-cytomegalovirus (CMV) antibodies reduce the incidence of CMV transmission and ameliorate the severity of CMV-associated disease. Neutralizing activity, measured as the ability of antibodies to prevent entry of cell-free virus, is an important component of natural immunity. However, in vivo CMV amplification may occur mainly via spread between adjacent cells within tissues. Thus, inhibition of cell-to-cell spread may be important when evaluating therapeutic antibodies or humoral responses to infection or immunization. In vitro CMV cell-to-cell spread is largely resistant to antibodies in fibroblast cultures but sensitive in endothelial cell cultures. In the present study antibodies in CMV hyperimmuneglobulin or seropositive human sera inhibited CMV cell-to-cell spread in epithelial cell cultures. Spread inhibition activity was quantitated with a GFP reporter assay employing GFP-tagged epithelialtropic variants of CMV strains Towne or AD169. Measurement of spread inhibition provides an additional parameter for the evaluation of candidate vaccines or immunotherapeutics and to further characterize the role of antibodies in controlling CMV transmission and disease. PMID:23669101

  20. Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells.

    PubMed

    Kassmer, Susannah H; Bruscia, Emanuela M; Zhang, Ping-Xia; Krause, Diane S

    2012-03-01

    Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM. PMID:22162244

  1. Alveolar epithelial cells orchestrate DC function in murine viral pneumonia

    PubMed Central

    Unkel, Barbara; Hoegner, Katrin; Clausen, Bjrn E.; Lewe-Schlosser, Peter; Bodner, Johannes; Gattenloehner, Stefan; Janen, Hermann; Seeger, Werner; Lohmeyer, Juergen; Herold, Susanne

    2012-01-01

    Influenza viruses (IVs) cause pneumonia in humans with progression to lung failure. Pulmonary DCs are key players in the antiviral immune response, which is crucial to restore alveolar barrier function. The mechanisms of expansion and activation of pulmonary DC populations in lung infection remain widely elusive. Using mouse BM chimeric and cell-specific depletion approaches, we demonstrated that alveolar epithelial cell (AEC) GM-CSF mediates recovery from IV-induced injury by affecting lung DC function. Epithelial GM-CSF induced the recruitment of CD11b+ and monocyte-derived DCs. GM-CSF was also required for the presence of CD103+ DCs in the lung parenchyma at baseline and for their sufficient activation and migration to the draining mediastinal lymph nodes (MLNs) during IV infection. These activated CD103+ DCs were indispensable for sufficient clearance of IVs by CD8+ T cells and for recovery from IV-induced lung injury. Moreover, GM-CSF applied intratracheally activated CD103+ DCs, inducing increased migration to MLNs, enhanced viral clearance, and attenuated lung injury. Together, our data reveal that GM-CSFdependent cross-talk between IV-infected AECs and CD103+ DCs is crucial for effective viral clearance and recovery from injury, which has potential implications for GM-CSF treatment in severe IV pneumonia. PMID:22996662

  2. Apical protein transport and lumen morphogenesis in polarized epithelial cells

    PubMed Central

    WILLENBORG, Carly; PREKERIS, Rytis

    2014-01-01

    Synopsis Segregation of the apical and basolateral plasma membrane domains is the key distinguishing feature of epithelial cells. A series of interrelated cues and processes follow this primary polarization event, resulting in the morphogenesis of the mammalian epithelium. This review focuses on the role of the interactions between the extracellular matrix and neighbouring cells during the initiation and establishment of epithelial polarity, and the role that membrane transport and polarity complexes play in this process. An overview of the formation of the apical junctional complexes is given in relation to the generation of distinct membrane domains characterized by the asymmetric distribution of phosphoinositides and proteins. The mechanisms and machinery utilized by the trafficking pathways involved in the generation and maintenance of this apical-basolateral polarization are expounded, highlighting processes of apical-directed transport. Furthermore, the current proposed mechanisms for the organization of entire networks of cells into a structured, polarized three-dimensional structure are described, with an emphasis on the proposed mechanisms for the formation and expansion of the apical lumen. PMID:21366541

  3. Growth factor responsiveness of human retinal pigment epithelial cells.

    PubMed

    Leschey, K H; Hackett, S F; Singer, J H; Campochiaro, P A

    1990-05-01

    Growth factor effects on DNA synthesis in density-arrested human retinal pigment epithelial cells were assessed by [3H]-thymidine incorporation. Acidic and basic fibroblast growth factor and epidermal growth factor were potent stimulators, whereas platelet-derived growth factor, insulinlike growth factor-1, and insulin were weak or modest stimulators when used alone. When used in combination, each of the above growth factors caused a significant enhancement of [3H]-thymidine incorporation regardless of its effect when used alone. The combination of all four growth factors was significantly more effective than all other combinations, demonstrating synergism in their action. Similar results were found in cell proliferation assays. In contrast to this, transforming growth factor-beta inhibited the ability of each of the other growth factors and serum-containing media to stimulate [3H]-thymidine incorporation. These data suggest that DNA synthesis in human retinal pigment epithelial cells can be modulated by several growth factors, some in a stimulatory or synergistic manner and at least one in an inhibitory manner. A better understanding of these complex interactions may provide insights relevant to normal and abnormal ocular wound healing. PMID:2186011

  4. Thrombin is a stimulator of retinal pigment epithelial cell proliferation.

    PubMed

    Hackett, S F; Singer, J H; Leschey, K H; Campochiaro, P A

    1991-07-01

    Two preparations of bovine thrombin were found to stimulate DNA synthesis in cultured human retinal pigment epithelial cells. DNA synthesis was assessed by both [3H]thymidine incorporation into TCA precipitable material and nuclear labeling with [3H]thymidine. Cultures grown in the presence of thrombin for 48 hr showed a significant increase in cell number. When the concentrations of the two thrombin preparations were normalized for clotting activity, they had almost identical dose-response curves and both caused a tenfold maximal stimulation of [3H]thymidine incorporation. The EC50 for the preparation with higher specific activity was 20 ng ml(-1). Hirudin, a specific high affinity inhibitor of thrombin, completely blocked the mitogenic effect. When a maximally effective concentration of thrombin was used in combination with maximally effective concentrations of other growth factors (insulin, acidic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor), they were found to be strongly synergistic in stimulating DNA synthesis. These data suggest that thrombin may act as an endocrine mediator of retinal pigment epithelial cell proliferation and participate in normal and exaggerated ocular wound healing. PMID:1879507

  5. DUSP10 regulates intestinal epithelial cell growth and colorectal tumorigenesis.

    PubMed

    Png, C W; Weerasooriya, M; Guo, J; James, S J; Poh, H M; Osato, M; Flavell, R A; Dong, C; Yang, H; Zhang, Y

    2016-01-14

    Dual specificity phosphatase 10 (DUSP10), also known as MAP kinase phosphatase 5 (MKP5), negatively regulates the activation of MAP kinases. Genetic polymorphisms and aberrant expression of this gene are associated with colorectal cancer (CRC) in humans. However, the role of DUSP10 in intestinal epithelial tumorigenesis is not clear. Here, we showed that DUSP10 knockout (KO) mice had increased intestinal epithelial cell (IEC) proliferation and migration and developed less severe colitis than wild-type (WT) mice in response to dextran sodium sulphate (DSS) treatment, which is associated with increased ERK1/2 activation and Krppel-like factor 5 (KLF5) expression in IEC. In line with increased IEC proliferation, DUSP10 KO mice developed more colon tumours with increased severity compared with WT mice in response to administration of DSS and azoxymethane (AOM). Furthermore, survival analysis of CRC patients demonstrated that high DUSP10 expression in tumours was associated with significant improvement in survival probability. Overexpression of DUSP10 in Caco-2 and RCM-1 cells inhibited cell proliferation. Our study showed that DUSP10 negatively regulates IEC growth and acts as a suppressor for CRC. Therefore, it could be targeted for the development of therapies for colitis and CRC. PMID:25772234

  6. Electronic device for microelectrode recordings in epithelial cells.

    PubMed

    Garcia-Diaz, J F; Stump, S; Armstrong, W M

    1984-03-01

    A device is described that permits continuous measurement of electrophysiological parameters in epithelial tissues in the open-circuit mode. Transepithelial potential (VT) and microelectrode (either conventional or ion-selective) potential (VM) are directly measured. Application of transepithelial current pulses allows continuous monitoring of transepithelial resistance (RT) and the ratio between the changes in VM and VT induced by these pulses. Measurement of this ratio, which under some circumstances reflects the apical fractional resistance of the cellular pathway, is important in assessing membrane damage during microelectrode impalement and/or as an index that the microelectrode tip is inside a cell. This is particularly useful when the change in VM during impalement is small. Application of 0.5-nA current pulses through open-tip microelectrodes allows continuous recording of the microelectrode resistance (RM). In epithelia where the individual cells are electrically coupled this permits acceptable impalements (RM remains nearly constant) to be distinguished from those affected by tip potential artifacts due to plugging of the microelectrode tip (RM increases after penetration of the cell membrane). The device provides compensation for the IR voltage drop in the solution between the potential measuring salt bridges and the epithelial surfaces. The microelectrode electrometer has an input impedance greater than 10(15) and is provided with stray capacitance neutralization. PMID:6703048

  7. Proteomic Analysis of Nasal Epithelial Cells from Cystic Fibrosis Patients

    PubMed Central

    Papon, Jean-Franois; Chhuon, Cerina; Zadigue, Patricia; Prulire-Escabasse, Virginie; Amselem, Serge; Escudier, Estelle; Coste, Andr; 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

  8. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

    PubMed Central

    2009-01-01

    Background Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. Results The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. Conclusion Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide. PMID:19650888

  9. Fas (CD95) Induces Alveolar Epithelial Cell Apoptosis in Vivo

    PubMed Central

    Matute-Bello, Gustavo; Winn, Robert K.; Jonas, Mechthild; Chi, Emil Y.; Martin, Thomas R.; Liles, W. Conrad

    2001-01-01

    Activation of the Fas/FasL system induces apoptosis of susceptible cells, but may also lead to nuclear factor ?B activation. Our goal was to determine whether local Fas activation produces acute lung injury by inducing alveolar epithelial cell apoptosis and by generating local inflammatory responses. Normal mice (C57BL/6) and mice deficient in Fas (lpr) were treated by intranasal instillation of the Fas-activating monoclonal antibody (mAb) Jo2 or an irrelevant control mAb, and studied 6 or 24 hours later using bronchoalveolar lavage (BAL), histopathology, DNA nick-end-labeling assays, and electron microscopy. Normal mice treated with mAb Jo2 had significant increases in BAL protein at 6 hours, and BAL neutrophils at 24 hours, as compared to lpr mice and to mice treated with the irrelevant mAb. Neutrophil recruitment was preceded by increased mRNA expression for tumor necrosis factor-?, macrophage inflammatory protein-1?, macrophage inflammatory protein-2, macrophage chemotactic protein-1, and interleukin-6, but not interferon-?, transforming growth factor-?, RANTES, eotaxin, or IP-10. Lung sections from Jo2-treated normal mice showed neutrophilic infiltrates, alveolar septal thickening, hemorrhage, and terminal dUTP nick-end-labeling-positive cells in the alveolar septae and airspaces. Type II pneumocyte apoptosis was confirmed by electron microscopy. Fas activation in vivo results in acute alveolar epithelial injury and lung inflammation, and may be important in the pathogenesis of acute lung injury. PMID:11141488

  10. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing

    PubMed Central

    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. PMID:26673160

  11. 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. PMID:26673160

  12. Production of arachidonic and linoleic acid metabolites by guinea pig tracheal epithelial cells

    SciTech Connect

    Oosthuizen, M.J.; Engels, F.; Van Esch, B.; Henricks, P.A.; Nijkamp, F.P. )

    1990-08-01

    Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B4 and C4 and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed.

  13. 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

  14. Normal and abnormal epithelial differentiation in the female reproductive tract.

    PubMed

    Kurita, Takeshi

    2011-10-01

    In mammals, the female reproductive tract (FRT) develops from a pair of paramesonephric or Mllerian ducts (MDs), which arise from coelomic epithelial cells of mesodermal origin. During development, the MDs undergo a dynamic morphogenetic transformation from simple tubes consisting of homogeneous epithelium and surrounding mesenchyme into several distinct organs namely the oviduct, uterus, cervix and vagina. Following the formation of anatomically distinctive organs, the uniform MD epithelium (MDE) differentiates into diverse epithelial cell types with unique morphology and functions in each organ. Classic tissue recombination studies, in which the epithelium and mesenchyme isolated from the newborn mouse FRT were recombined, have established that the organ specific epithelial cell fate of MDE is dictated by the underlying mesenchyme. The tissue recombination studies have also demonstrated that there is a narrow developmental window for the epithelial cell fate determination in MD-derived organs. Accordingly, the developmental plasticity of epithelial cells is mostly lost in mature FRT. If the signaling that controls epithelial differentiation is disrupted at the critical developmental stage, the cell fate of MD-derived epithelial tissues will be permanently altered and can result in epithelial lesions in adult life. A disruption of signaling that maintains epithelial cell fate can also cause epithelial lesions in the FRT. In this review, the pathogenesis of cervical/vaginal adenoses and uterine squamous metaplasia is discussed as examples of such incidences. PMID:21612855

  15. Normal and Abnormal Epithelial Differentiation in the Female Reproductive Tract

    PubMed Central

    Kurita, Takeshi

    2011-01-01

    In mammals, the female reproductive tract (FRT) develops from a pair of paramesonephric or Mllerian ducts (MDs), which arise from coelomic epithelial cells of mesodermal origin. During development, the MDs undergo a dynamic morphogenetic transformation from simple tubes consisting of homogeneous epithelium and surrounding mesenchyme into several distinct organs namely the oviduct, uterus, cervix and vagina. Following the formation of anatomically distinctive organs, the uniform MD epithelium (MDE) differentiates into diverse epithelial cell types with unique morphology and functions in each organ. Classic tissue recombination studies, in which the epithelium and mesenchyme isolated from the newborn mouse FRT were recombined, have established that the organ specific epithelial cell fate of MDE is dictated by the underlying mesenchyme. The tissue recombination studies have also demonstrated that there is a narrow developmental window for the epithelial cell fate determination in MD-derived organs. Accordingly, the developmental plasticity of epithelial cells is mostly lost in mature FRT. If the signaling that controls epithelial differentiation is disrupted at the critical developmental stage, the cell fate of MD-derived epithelial tissues will be permanently altered and can result in epithelial lesions in adult life. A disruption of signaling that maintains epithelial cell fate can also cause epithelial lesions in the FRT. In this review, the pathogenesis of cervical/vaginal adenoses and uterine squamous metaplasia is discussed as examples of such incidences. PMID:21612855

  16. 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. PMID:24500281

  17. PKC?/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)? 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 PKC?-dependent pathway and hypothesized that PKC?-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 PKC?. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKC? from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKC?, revealed that PKC? 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, ?-smooth muscle actin, and vimentin. We conclude that PKC?/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKC? and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases. PMID:24500281

  18. Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition

    PubMed Central

    Wang, Wenyi; Kato, Satomi; Colvocoresses-Dodds, Jennifer; Fifadara, Nimita H.; Gauthier, Theresa W.; Helms, My N.; Carlton, David P.; Brown, Lou Ann S.

    2013-01-01

    Myofibroblast accumulation is a pathological feature of lung diseases requiring oxygen therapy. One possible source for myofibroblasts is through the epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AEC). To study the effects of oxygen on alveolar EMT, we used RLE-6TN and ex vivo lung slices and found that hyperoxia (85% O2, H85) decreased epithelial proteins, presurfactant protein B (pre-SpB), pro-SpC, and lamellar protein by 50% and increased myofibroblast proteins, ?-smooth muscle actin (?-SMA), and vimentin by over 200% (P < 0.05). In AEC freshly isolated from H85-treated rats, mRNA for pre-SpB and pro-SpC was diminished by ?50% and ?-SMA was increased by 100% (P < 0.05). Additionally, H85 increased H2O2 content, and H2O2 (2550 ?M) activated endogenous transforming growth factor-?1 (TGF-?1), as evident by H2DCFDA immunofluorescence and ELISA (P < 0.05). Both hyperoxia and H2O2 increased SMAD3 phosphorylation (260% of control, P < 0.05). Treating cultured cells with TGF-?1 inhibitors did not prevent H85-induced H2O2 production but did prevent H85-mediated ?-SMA increases and E-cadherin downregulation. Finally, to determine the role of TGF-?1 in hyperoxia-induced EMT in vivo, we evaluated AEC from H85-treated rats and found that vimentin increased ?10-fold (P < 0.05) and that this effect was prevented by intraperitoneal TGF-?1 inhibitor SB-431542. Additionally, SB-431542 treatment attenuated changes in alveolar histology caused by hyperoxia. Our studies indicate that hyperoxia promotes alveolar EMT through a mechanism that is dependent on activation of TGF-?1 signaling. PMID:24375795

  19. Magnetite induces oxidative stress and apoptosis in lung epithelial cells.

    PubMed

    Ramesh, Vani; Ravichandran, Prabakaran; Copeland, Clinton L; Gopikrishnan, Ramya; Biradar, Santhoshkumar; Goornavar, Virupaxi; Ramesh, Govindarajan T; Hall, Joseph C

    2012-04-01

    There is an ongoing concern regarding the biocompatibility of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. In this study, we investigated the toxic properties of magnetite stabilized with polyacrylate sodium. The magnetite was characterized by X-ray powder diffraction analysis, and the mean particle diameter was calculated using the Scherrer formula and was found to be 9.3 nm. In this study, we treated lung epithelial cells with different concentrations of magnetite and investigated their effects on oxidative stress and cell proliferation. Our data showed an inhibition of cell proliferation in magnetite-treated cells with a significant dose-dependent activation and induction of reactive oxygen species. Also, we observed a depletion of antioxidants, glutathione, and superoxide dismutase, respectively, as compared with control cells. In addition, apoptotic-related protease/enzyme such as caspase-3 and -8 activities, were increased in a dose-dependent manner with corresponding increased levels of DNA fragmentation in magnetite-treated cells compared to than control cells. Together, the present study reveals that magnetite exposure induces oxidative stress and depletes antioxidant levels in the cells to stimulate apoptotic pathway for cell death. PMID:22147200

  20. Effect of curcumin on aging retinal pigment epithelial cells

    PubMed Central

    Zhu, Wei; Wu, Yan; Meng, Yi-Fang; Wang, Jin-Yu; Xu, Ming; Tao, Jian-Jun; Lu, Jiong

    2015-01-01

    Age-related macular degeneration (AMD) is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE) cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 M, 40 M, and 80 M. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE cell model. Curcumin improved cell viability and decreased apoptosis and oxidative stress. Further, curcumin had a significant influence on expression of apoptosis-associated proteins and oxidative stress biomarkers. In conclusion, treatment with curcumin was able to regulate proliferation, oxidative stress, and apoptosis in aging RPE cells. Accordingly, application of curcumin may be a novel strategy to protect against age-related change in AMD. PMID:26445530

  1. Effect of curcumin on aging retinal pigment epithelial cells.

    PubMed

    Zhu, Wei; Wu, Yan; Meng, Yi-Fang; Wang, Jin-Yu; Xu, Ming; Tao, Jian-Jun; Lu, Jiong

    2015-01-01

    Age-related macular degeneration (AMD) is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE) cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 M, 40 M, and 80 M. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE cell model. Curcumin improved cell viability and decreased apoptosis and oxidative stress. Further, curcumin had a significant influence on expression of apoptosis-associated proteins and oxidative stress biomarkers. In conclusion, treatment with curcumin was able to regulate proliferation, oxidative stress, and apoptosis in aging RPE cells. Accordingly, application of curcumin may be a novel strategy to protect against age-related change in AMD. PMID:26445530

  2. Interleukin-7 Links T Lymphocyte and Intestinal Epithelial Cell Homeostasis

    PubMed Central

    Shalapour, Shabnam; Deiser, Katrin; Khl, Anja A.; Glauben, Rainer; Krug, Susanne M.; Fischer, Andr; Sercan, zen; Chappaz, Stephane; Bereswill, Stefan; Heimesaat, Markus M.; Loddenkemper, Christoph; Fromm, Michael; Finke, Daniela; Hmmerling, Gnter J.; Arnold, Bernd; Siegmund, Britta; Schler, Thomas

    2012-01-01

    Interleukin-7 (IL-7) is a major survival factor for mature T cells. Therefore, the degree of IL-7 availability determines the size of the peripheral T cell pool and regulates T cell homeostasis. Here we provide evidence that IL-7 also regulates the homeostasis of intestinal epithelial cells (IEC), colon function and the composition of the commensal microflora. In the colon of T cell-deficient, lymphopenic mice, IL-7-producing IEC accumulate. IEC hyperplasia can be blocked by IL-7-consuming T cells or the inactivation of the IL-7/IL-7R signaling pathway. However, the blockade of the IL-7/IL-7R signaling pathway renders T cell-deficient mice more sensitive to chemically-induced IEC damage and subsequent colitis. In summary, our data demonstrate that IL-7 promotes IEC hyperplasia under lymphopenic conditions. Under non-lymphopenic conditions, however, T cells consume IL-7 thereby limiting IEC expansion and survival. Hence, the degree of IL-7 availability regulates both, T cell and IEC homeostasis. PMID:22384106

  3. Kidney injury molecule–1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells

    PubMed Central

    Ichimura, Takaharu; Asseldonk, Edwin J.P.v.; Humphreys, Benjamin D.; Gunaratnam, Lakshman; Duffield, Jeremy S.; Bonventre, Joseph V.

    2008-01-01

    Following injury, the clearance of apoptotic and necrotic cells is necessary for mitigation and resolution of inflammation and tissue repair. In addition to macrophages, which are traditionally assigned to this task, neighboring epithelial cells in the affected tissue are postulated to contribute to this process. Kidney injury molecule–1 (KIM-1 or TIM-1) is an immunoglobulin superfamily cell-surface protein not expressed by cells of the myeloid lineage but highly upregulated on the surface of injured kidney epithelial cells. Here we demonstrate that injured kidney epithelial cells assumed attributes of endogenous phagocytes. Confocal images confirm internalization of apoptotic bodies within KIM-1–expressing epithelial cells after injury in rat kidney tubules in vivo. KIM-1 was directly responsible for phagocytosis in cultured primary rat tubule epithelial cells and also porcine and canine epithelial cell lines. KIM-1 was able to specifically recognize apoptotic cell surface-specific epitopes phosphatidylserine, and oxidized lipoproteins, expressed by apoptotic tubular epithelial cells. Thus, KIM-1 is the first nonmyeloid phosphatidylserine receptor identified to our knowledge that transforms epithelial cells into semiprofessional phagocytes. PMID:18414680

  4. WU Polyomavirus in Respiratory Epithelial Cells from Lung Transplant Patient with Job Syndrome

    PubMed Central

    Siebrasse, Erica A.; Pastrana, Diana V.; Nguyen, Nang L.; Wang, Annie; Roth, Mark J.; Holland, Steven M.; Freeman, Alexandra F.; McDyer, John; Buck, Christopher B.

    2015-01-01

    We detected WU polyomavirus (WUPyV) in a bronchoalveolar lavage sample from lungs transplanted into a recipient with Job syndrome by using immunoassays specific for the WUPyV viral protein 1. Co-staining for an epithelial cell marker identified most WUPyV viral protein 1–positive cells as respiratory epithelial cells. PMID:25531075

  5. ISOLATION AND LONG-TERM CULTURE OF RAT, RABBIT, AND HUMAN NASAL TURBINATE EPITHELIAL CELLS

    EPA Science Inventory

    Nasal turbinate epithelial cells were isolated from rats, rabbits, and humans using either a surgical or an in situ enzyme incubation technique. The culture conditions that permit optimal cell attachment and selective growth of the nasal epithelial cells were determined. These co...

  6. Glycerol monolaurate does not alter rhesus macaque (Macaca mulatta) vaginal lactobacilli and is safe for chronic use.

    PubMed

    Schlievert, Patrick M; Strandberg, Kristi L; Brosnahan, Amanda J; Peterson, Marnie L; Pambuccian, Stefan E; Nephew, Karla R; Brunner, Kevin G; Schultz-Darken, Nancy J; Haase, Ashley T

    2008-12-01

    Glycerol monolaurate (GML) is a fatty acid monoester that inhibits growth and exotoxin production of vaginal pathogens and cytokine production by vaginal epithelial cells. Because of these activities, and because of the importance of cytokine-mediated immune activation in human immunodeficiency virus type 1 (HIV-1) transmission to women, our laboratories are performing studies on the potential efficacy of GML as a topical microbicide to interfere with HIV-1 transmission in the simian immunodeficiency virus-rhesus macaque model. While GML is generally recognized as safe by the FDA for topical use, its safety for chronic use and effects on normal vaginal microflora in this animal model have not been evaluated. GML was therefore tested both in vitro for its effects on vaginal flora lactobacilli and in vivo as a 5% gel administered vaginally to monkeys. In vitro studies demonstrated that lactobacilli are not killed by GML; GML blocks the loss of their viability in stationary phase and does not interfere with lactic acid production. GML (5% gel) does not quantitatively alter monkey aerobic vaginal microflora compared to vehicle control gel. Lactobacilli and coagulase-negative staphylococci are the dominant vaginal aerobic microflora, with beta-hemolytic streptococci, Staphylococcus aureus, and yeasts sporadically present; gram-negative rods are not part of their vaginal flora. Colposcopy and biopsy studies indicate that GML does not alter normal mucosal integrity and does not induce inflammation; instead, GML reduces epithelial cell production of interleukin 8. The studies suggest that GML is safe for chronic use in monkeys when applied vaginally; it does not alter either mucosal microflora or integrity. PMID:18838587

  7. Stepwise Protocol for Cytospin-enhanced Smearing for Scraped Corneal Epithelial Cells.

    PubMed

    Jeyalatha, Mani V; Malathi, Jambulingam; Madhavan, Hajib N

    2016-01-01

    Proteins and antigens present on the cell surface are usually determined by immunofluorescence staining. Uniform distribution of cells is required to appreciate the presence of surface proteins. Improper smearing or crushing of the corneal epithelial cells can potentially destroy the cellular integrity. Thus a simplified, systemic method was designed to smear the cells scraped from the cornea. The procedure includes trypsinisation for dissociation of corneal epithelial cells and cytospinning for concentrating the cells in a smear. The standardized protocol was found to be efficient in maintaining the integrity of the corneal epithelial cells and also the distribution of the cells in the smear. PMID:26633702

  8. Hormonal factors in vaginal candidiasis in rats.

    PubMed Central

    Kinsman, O S; Collard, A E

    1986-01-01

    The hormonal status of rats affected vaginal infection with Candida albicans. Four hours after infection viable counts were higher and germ tubes were longer in those animals in estrous than in other animals. However, the infection was not maintained with the change in epithelial cell type which occurred as part of the estrous cycle. Estrogen dosing following ovariectomy predisposed toward infection, while progesterone dosing did not. In rats injected with progesterone, germ tube clumping was seen, leukocytes were present, and elimination occurred before hyphal growth was evident. In rats injected with estrogen, however, infection was maintained, with hyphal growth extending throughout the cornified epithelial layer. Vaginal washings from rats dosed with estrogen promoted elongation of germ tubes in vitro to a greater extent than washings from other rats. Preincubation of blastospores in progesterone and subsequent infection of rats in pseudoestrous promoted clumping of germ tubes in the vagina. Strains of C. albicans varied in their virulence, which correlated with their ability to produce germ tubes in vitro. Loss of virulence occurred on subculture of a clinical isolate. Images PMID:3527984

  9. Mapping the dynamics of force transduction at cell-cell junctions of epithelial clusters.

    PubMed

    Ng, Mei Rosa; Besser, Achim; Brugge, Joan S; Danuser, Gaudenz

    2014-01-01

    Force transduction at cell–cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both sub-cellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell–cell junctions. At the multi-cellular scale, cell–cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell–cell adhesions [corrected]. PMID:25479385

  10. Efficient T cell repertoire selection in tetraparental chimeric mice independent of thymic epithelial MHC

    PubMed Central

    Martinic, Marianne M.; Rlicke, Thomas; Althage, Alana; Odermatt, Bernhard; Hchli, Matthias; Lamarre, Alain; Dumrese, Tilman; Speiser, Daniel E.; Kyburz, Diego; Hengartner, Hans; Zinkernagel, Rolf M.

    2003-01-01

    Nonthymic epithelial cells were compared with thymic epithelial cells for their role in T cell repertoire selection. Tetraparental aggregation chimeras were generated from T and B cell-deficient mice (H-2d SCID or H-2b Rag?/?) and thymus-deficient nude mice (H-2b or H-2d). These tetraparental mice showed primary protective CD8+ T cell responses, after lymphocytic choriomeningitis virus infection, that were peptide-specifically restricted to either thymic or nonthymic epithelial MHC at comparable levels. These chimeras also mounted neutralizing IgG responses dependent on cognate CD4+ T helper cell activity restricted to nonthymic epithelial MHC. Therefore, in contrast to earlier results with irradiation or thymus chimeras, these relatively undisturbed tetraparental mice reveal that the MHC of nonthymic epithelial cells efficiently selects a functional T cell repertoire. PMID:12574503

  11. Group B Streptococcus CovR regulation modulates host immune signaling pathways to promote vaginal colonization

    PubMed Central

    Patras, Kathryn A.; Wang, Nai-Yu; Fletcher, Erin M.; Cavaco, Courtney K.; Jimenez, Alyssa; Garg, Mansi; Fierer, Joshua; Sheen, Tamsin R.; Rajagopal, Lakshmi; Doran, Kelly S.

    2013-01-01

    Summary Streptococcus agalactiae (Group B Streptococcus, GBS) is a frequent commensal organism of the vaginal tract of healthy women. However, GBS can transition to a pathogen in susceptible hosts, but host and microbial factors that contribute to this conversion are not well understood. GBS CovR/S (CsrR/S) is a two component regulatory system that regulates key virulence elements including adherence and toxin production. We performed global transcription profiling of human vaginal epithelial cells exposed to WT, CovR deficient, and toxin deficient strains, and observed that insufficient regulation by CovR and subsequent increased toxin production results in a drastic increase in host inflammatory responses, particularly in cytokine signaling pathways promoted by IL-8 and CXCL2. Additionally, we observed that CovR regulation impacts epithelial cell attachment and intracellular invasion. In our mouse model of GBS vaginal colonization, we further demonstrated that CovR regulation promotes vaginal persistence, as infection with a CovR deficient strain resulted in a heightened host immune response as measured by cytokine production and neutrophil activation. Using CXCr2 KO mice, we determined that this immune alteration occurs, at least in part, via signaling through the CXCL2 receptor. Taken together, we conclude that CovR is an important regulator of GBS vaginal colonization and loss of this regulatory function may contribute to the inflammatory havoc seen during the course of infection. PMID:23298320

  12. Equine tracheal epithelial membrane strips - An alternate method for examining epithelial cell arachidonic acid metabolism

    SciTech Connect

    Gray, P.R.; Derksen, F.J.; Robinson, N.E.; Peter-Golden, M.L. Univ. of Michigan, Ann Arbor )

    1990-02-26

    Arachidonic acid metabolism by tracheal epithelium can be studied using enzymatically dispersed cell suspensions or cell cultures. Both techniques require considerable tissue disruption and manipulation and may not accurately represent in vivo activity. The authors have developed an alternate method for obtaining strips of equine tracheal epithelium without enzymatic digestion. In the horse, a prominent elastic lamina supports the tracheal epithelium. By physical splitting this lamina, they obtained strips ({le}12 x 1.5 cm) of pseudostratified columnar epithelium attached to a layer of elastic tissue 30-100 {mu}m thick. Epithelial strips (1.2 x 0.5 cm) were attached to plexiglass rods and incubated with ({sup 3}H)arachidonic acid in M199 medium (0.5 {mu}Ci/ml) for 24 hours at 37C. The strips incorporated 36{+-}4% (mean {+-} SEM) of the total radioactivity and released 8.0{+-}1.2% of incorporated radioactivity when stimulated by 5.0 {mu}M calcium ionophore A23187. The extracted supernatant was processed using HPLC, resulting in peaks of radioactivity that co-eluted with authentic PGE{sub 2}, PGF{sub 2}{alpha}, and 12-HETE standards. The greatest activity corresponded to the PGE{sub 2} and PGF{sub 2}{alpha} standards, which is a similar pattern to that reported for cultured human tracheal epithelium.

  13. NK cells are necessary for recovery of corneal CD11c+ dendritic cells after epithelial abrasion injury

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mechanisms controlling CD11c(+) MHCII(+) DCs during corneal epithelial wound healing were investigated in a murine model of corneal abrasion. Selective depletion of NKp46(+) CD3- NK cells that normally migrate into the cornea after epithelial abrasion resulted in >85% reduction of the epithelial CD1...

  14. Prevalence of vaginal infections and associated lifestyles of students in the university of Cape Coast, Ghana

    PubMed Central

    Aubyn, Gloria Baaba; Tagoe, Daniel Nii Aryee

    2013-01-01

    Objective To determine the prevalence of vaginal infections and associated lifestyles of students visiting the University of Cape Coast Hospital. Methods Fifty female students presenting with clinical symptoms of vaginitis were sampled. One hundred samples made up of 50 urine and 50 higher vaginal swabs (HVS) were obtained from patients and questionnaire administered. Samples were wet prepared, examined microscopically, and cultured on blood and chocolate agars for 24 h at (352) C. Colonial morphology, Gram reactions and biochemical tests were used for the identification of isolates. Results There were high percentages of pus cells (64%), epithelial cells (62%) and yeast cells (56%) in all urine samples. Bacterial isolates included Staphylococcus aureus (28%) and (22%), Klebsiella spp. (6%) and (4%) in urine and HVS samples respectively; Escherichia coli in urine (18%) and Candida in HVS (16%). The overall prevalence of vaginitis was 66%, including bacterial vaginosis 28%, Candida infection 22% and co-infection of bacterial and Candida 16%. Lifestyle data showed all sampled students were sexually active, 48% used contraceptives, 54% used antimicrobial agents, and 92% prefered wearing of trousers and shorts. Conclusions The present study indicates prevalence of vaginal infection among female students, which strongly correlates with student lifestyle. Education on lifestyle modifications will go a long way in reducing the prevalence of vaginitis.

  15. 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 laboratory—are cultured in relevant 3D microenvironments. We focus on the design of functional assays that enable us to understand the intricate signaling events underlying mammary gland biology, and address the advantages and limitations of the different culture settings. Finally we also discuss how advances in bioengineering tools may help towards the ultimate goal of building tissues and organs in culture for basic research and clinical studies. PMID:23097110

  16. Specific forms of BAFF favor BAFF receptor-mediated epithelial cell survival.

    PubMed

    Lahiri, Ayan; Varin, Marie-Michle; Le Pottier, Latitia; Pochard, Pierre; Bendaoud, Boutahar; Youinou, Pierre; Pers, Jacques-Olivier

    2014-06-01

    Although B cell activating factor (BAFF) and its receptor BR3 are produced and expressed by many cells, their role has been restricted to the lymphocyte lineage. Using various techniques (RT-PCR, indirect immunofluorescence, flow cytometry analysis), we observed the expression of BR3 and the production of BAFF by the human salivary gland cell line, by epithelial cells from biopsies of Sjgren's syndrome patients and their controls, but also by salivary gland epithelial cells in culture. To decipher the role of BAFF and BR3 on epithelial cells, BAFF and BR3 were neutralized by blocking antibodies or RNA specific inhibitor (siBR3) and epithelial cell survival was analyzed. Blocking BR3 promotes epithelial cell apoptosis invitro. This apoptosis resulted in the nuclear translocation of PKC?. BAFF neutralization by various anti-BAFF antibodies leads to different effects depending on the antibody used suggesting that only some forms of BAFF are required for epithelial cell survival. Our study demonstrates that BR3 is involved in the survival of cultured epithelial cells due to an autocrine effect of BAFF. It also suggests that epithelial cells produce different forms of BAFF and that only some of them are responsible for this effect. PMID:24602383

  17. Confluent monolayers of bile duct epithelial cells with tight junctions.

    PubMed

    Okamoto, H; Ishii, M; Mano, Y; Igarashi, T; Ueno, Y; Kobayashi, K; Toyota, T

    1995-07-01

    The culture of fully differentiated intrahepatic bile duct epithelial cells (IBDECs) to use as a model for the in vivo intrahepatic biliary tract has not been established. IBDECs from normal rat livers were grown on a collagen-coated permeable filter and formed a confluent monolayer 7 days after being plated. Positive reactions for cytokeratin-19 and retained gamma-glutamyl transpeptidase (GGTP) activity were shown. The transepithelial electrical resistance between the apical and the basolateral compartment culture chambers increased with the culture age and plateaued after the 7th day. The resulting cultured cells displayed a number of characteristics. (1) The cells formed a thin, continuous monolayer and displayed microvilli on the apical surface and junctional complexes between the cells, consistent with in vivo IBDECs. (2) Cells cultured for more than 7 days prevented the passage of horseradish peroxidase (HRP) and ruthenium red through paracellular pathways. (3) Seven-day-old cultures displayed a mean transepithelial electrical resistance of 137.3 omega-cm2, which decreased by 27.1% from its initial level after cell treatment with ethylenediamineteraacetic acid (EDTA). These results indicate that confluent IBDEC monolayers are well differentiated and polarized with tight junctions (TJs) between the cells. These cell monolayers can provide a useful and relevant model for the in vitro study of various in vivo bile duct phenomena. PMID:7601408

  18. Efficient Generation of Functional Epithelial and Epidermal Cells from Human Pluripotent Stem Cells Under Defined Conditions

    PubMed Central

    Selekman, Joshua A.; Grundl, Nicholas J.; Kolz, Joshua M.

    2013-01-01

    Human pluripotent stem cells (hPSCs) have an unparalleled potential to generate limitless quantities of any somatic cell type. However, current methods for producing populations of various somatic cell types from hPSCs are generally not standardized and typically incorporate undefined cell culture components often resulting in variable differentiation efficiencies and poor reproducibility. To address this, we have developed a defined approach for generating epithelial progenitor and epidermal cells from hPSCs. In doing so, we have identified an optimal starting cell density to maximize yield and maintain high purity of K18+/p63+ simple epithelial progenitors. In addition, we have shown that the use of synthetic, defined substrates in lieu of Matrigel and gelatin can successfully facilitate efficient epithelial differentiation, maintaining a high (>75%) purity of K14+/p63+ keratinocyte progenitor cells and at a two to threefold higher yield than a previously reported undefined differentiation method. These K14+/p63+ cells also exhibited a higher expansion potential compared to cells generated using an undefined differentiation protocol and were able to terminally differentiate and recapitulate an epidermal tissue architecture in vitro. In summary, we have demonstrated the production of populations of functional epithelial and epidermal cells from multiple hPSC lines using a new, completely defined differentiation strategy. PMID:23560510

  19. New technique for culturing corneal epithelial cells of normal mice

    PubMed Central

    Kobayashi, Takeshi; Yoshioka, Ryuji; Ohashi, Yuichi

    2009-01-01

    Purpose To describe a new method of culturing mouse corneal epithelial cells (MCECs). Methods MCECs were isolated from C57/BL6 mouse corneas and cultured on type-I collagen-coated plastic dishes in low-calcium progenitor cell targeting medium (CnT-50). Expression of the mRNAs of N-terminal truncated isoform of p63 (DNp63), cytokeratin 12 (K12), and cytokeratin 14 (K14) were determined by reverse transcription-polymerase chain reaction (RT-PCR). To examine the differentiation capabilities, passage 3 (P3) MCECs at confluence were subcultured on amniotic membrane (AM) in a differentiation medium (CnT-30) until confluence. At confluence, 1 mM calcium was added and cultured for 4 more days. The expression of K12 in the stratified MCECs was analyzed by immunostaining. Results The MCECs cultured in CnT-50 proliferated until at least P10. The number of cells at confluency at P3 was 61.8 (SD 9.4, n=5) times that at P0. MCECs cultured on AM in CnT-30 with addition of calcium were stratified up to two to three layers, and the stratified MCECs expressed K12. DNp63 mRNA was continuously expressed throughout the different passages, and K12 mRNA was detected in P0 cells and the stratified MCECs on AM. Conclusions Cultured MCECs maintain their proliferation and differentiation capabilities as well as their corneal epithelial cell characteristics. These results suggest that MCECs produced by this culturing method provide a useful experimental model which can enable further development of research of the corneal epithelium. PMID:19693295

  20. Baicalin and baicalein inhibit transforming growth factor-?1-mediated epithelial-mesenchymal transition in human breast epithelial cells.

    PubMed

    Chung, Heesung; Choi, Hack Sun; Seo, Eun-Kyoung; Kang, Duk-Hee; Oh, Eok-Soo

    2015-03-13

    Since the epithelial-mesenchymal transition (EMT) is involved in many crucial functions of cancer cells, we set out to identify a natural compound capable of inhibiting EMT processes. TGF-?1 treatment induces EMT among normal mammary epithelial cells (MCF10A cells), as reflected by characteristic morphological changes into the fibroblastic phenotype, reduced expression of E-cadherin. Interestingly, butanol extracts of Scutellaria baicalensis Georgi significantly reduced the TGF-?1-mediated EMT of MCF10A cells. Further analysis revealed that baicalin and baicalein, the major flavones of these butanol extracts, inhibited TGF-?1-mediated EMT by reducing the expression level of the EMT-related transcription factor, Slug via the NF-?B pathway, and subsequently increased migration in MCF10A cells. Finally, both compounds reduced the TGF-?1-mediated EMT, anchorage-independent growth and cell migration of human breast cancer cells (MDA-MB-231 cells). Taken together, these results suggest that baicalin and baicalein of Scutellaria baicalensis Georgi may suppress the EMT of breast epithelial cells and the tumorigenic activity of breast cancer cells. Thus, these compounds could have potential as therapeutic or supplementary agents for the treatment of breast cancer. PMID:25686495

  1. Methods toward in vivo measurement of zebrafish epithelial and deep cell proliferation.

    PubMed

    Campana, Matteo; Maury, Benoit; Dutreix, Marie; Peyriras, Nadine; Sarti, Alessandro

    2010-05-01

    We present a strategy for automatic classification and density estimation of epithelial enveloping layer (EVL) and deep layer (DEL) cells, throughout zebrafish early embryonic stages. Automatic cells classification provides the bases to measure the variability of relevant parameters, such as cells density, in different classes of cells and is finalized to the estimation of effectiveness and selectivity of anticancer drug in vivo. We aim at approaching these measurements through epithelial/deep cells classification, epithelial area and thickness measurement, and density estimation from scattered points. Our procedure is based on Minimal Surfaces, Otsu clustering, Delaunay Triangulation, and Within-R cloud of points density estimation approaches. In this paper, we investigated whether the distance between nuclei and epithelial surface is sufficient to discriminate epithelial cells from deep cells. Comparisons of different density estimators, experimental results, and extensively accuracy measurements are included. PMID:19781805

  2. Cytomorphometric analysis of vaginal cells during normal cycle, under oral contraceptive therapy or in-vitro fertilization stimulation protocol.

    PubMed

    Chretien, M F; Lebouvier, B; Denis, A; Chappard, D

    1998-10-01

    A cytomorphometric analysis of superficial vaginal cells in women in three groups of different types of hormonal concentration was made. There were 15 women in each group. Group I was studied during a natural cycle, group II under oral contraceptive therapy and group III during an in-vitro fertilization (IVF) stimulation protocol. Morphometric parameters were measured on an image analyser. The area, perimeter and several form factors were measured separately for nuclei and cytoplasm. The nucleus:cytoplasmic ratio was also determined. The cytoplasmic area was significantly reduced in group II and was associated with a statistically significant reduction of the nuclear area. The nucleus:cytoplasmic ratio appeared significantly increased in group II and reduced in group III. Low oestradiol impregnation obtained with an oral minidosed contraceptive interfered with vaginal cell maturation. High oestradiol concentrations obtained during IVF protocols induced marked nuclear pycnosis but did not induce supra-physiological cell enlargement. Maximal cell size is genetically regulated according to Driesch's law of volume invariance and hormonal over-stimulation has no effect on cell size. The nucleus:cytoplasmic ratio appears to be a powerful parameter reflecting the opposite effects of hormones on cell compartments. PMID:9804228

  3. Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions.

    PubMed

    Short, Kirsty R; Kasper, Jennifer; van der Aa, Stijn; Andeweg, Arno C; Zaaraoui-Boutahar, Fatiha; Goeijenbier, Marco; Richard, Mathilde; Herold, Susanne; Becker, Christin; Scott, Dana P; Limpens, Ronald W A L; Koster, Abraham J; Bárcena, Montserrat; Fouchier, Ron A M; Kirkpatrick, Charles James; Kuiken, Thijs

    2016-03-01

    A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear.Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelial-endothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber.We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4.Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection. PMID:26743480

  4. Management of vaginitis.

    PubMed

    Owen, Marion K; Clenney, Timothy L

    2004-12-01

    Common infectious forms of vaginitis include bacterial vaginosis, vulvovaginal candidiasis, and trichomoniasis. Vaginitis also can occur because of atrophic changes. Bacterial vaginosis is caused by proliferation of Gardnerella vaginalis, Mycoplasma hominis, and anaerobes. The diagnosis is based primarily on the Amsel criteria (milky discharge, pH greater than 4.5, positive whiff test, clue cells in a wet-mount preparation). The standard treatment is oral metronidazole in a dosage of 500 mg twice daily for seven days. Vulvovaginal candidiasis can be difficult to diagnose because characteristic signs and symptoms (thick, white discharge, dysuria, vulvovaginal pruritus and swelling) are not specific for the infection. Diagnosis should rely on microscopic examination of a sample from the lateral vaginal wall (10 to 20 percent potassium hydroxide preparation). Cultures are helpful in women with recurrent or complicated vulvovaginal candidiasis, because species other than Candida albicans (e.g., Candida glabrata, Candida tropicalis) may be present. Topical azole and oral fluconazole are equally efficacious in the management of uncomplicated vulvovaginal candidiasis, but a more extensive regimen may be required for complicated infections. Trichomoniasis may cause a foul-smelling, frothy discharge and, in most affected women, vaginal inflammatory changes. Culture and DNA probe testing are useful in diagnosing the infection; examinations of wet-mount preparations have a high false-negative rate. The standard treatment for trichomoniasis is a single 2-g oral dose of metronidazole. Atrophic vaginitis results from estrogen deficiency. Treatment with topical estrogen is effective. PMID:15606061

  5. Depleted uranium induces neoplastic transformation in human lung epithelial cells.

    PubMed

    Xie, Hong; LaCerte, Carolyne; Thompson, W Douglas; Wise, John Pierce

    2010-02-15

    Depleted uranium (DU) is commonly used in military armor and munitions, and thus, exposure of soldiers and noncombatants is frequent and widespread. Previous studies have shown that DU has both chemical and radiological toxicity and that the primary route of exposure of DU to humans is through inhalation and ingestion. However, there is limited research information on the potential carcinogenicity of DU in human bronchial cells. Accordingly, we determined the neoplastic transforming ability of particulate DU to human bronchial epithelial cells (BEP2D). We observed the loss of contact inhibition and anchorage independent growth in cells exposed to DU after 24 h. We also characterized these DU-induced transformed cell lines and found that 40% of the cell lines exhibit alterations in plating efficiency and no significant changes in the cytotoxic response to DU. Cytogenetic analyses showed that 53% of the DU-transformed cell lines possess a hypodiploid phenotype. These data indicate that human bronchial cells are transformed by DU and exhibit significant chromosome instability consistent with a neoplastic phenotype. PMID:20000475

  6. Murine mechanical ventilation stimulates alveolar epithelial cell proliferation.

    PubMed

    Chess, Patricia Rose; Benson, Randi Potter; Maniscalco, William M; Wright, Terry W; O'Reilly, Michael A; Johnston, Carl J

    2010-08-01

    High tidal volume mechanical ventilation can cause inflammation and lung damage. Mechanical strain is also necessary for normal lung growth. The current work was performed to determine if mechanical ventilation with clinically utilized tidal volumes stimulates a proliferative response in the lung. Six- to 8-week-old C57/Bl6 mice, anesthetized with ketamine/xylozine, were ventilated for 6 hours with 10 mL/kg tidal volume, positive end-expiratory pressure (PEEP) 3cm H(2)O. Pulmonary function testing demonstrated decreased compliance within 3 hours of ventilation. Assessment of bronchoalveolar lavage (BAL) demonstrated no significant increase in lactate dehydrogenase, total lavagable cell number, or total protein after ventilation. There was evidence of inflammation in the lungs of ventilated mice, with an increased percentage of lymphocytes and neutrophils in BAL, and an increase in macrophage inflammatory protein (MIP)-2 and interleukin (IL)-1beta message in lung tissue. Immunohistochemistry of inflation-fixed lungs demonstrated increased alveolar cell proliferation, as measured by both proliferating cell nuclear antigen and Ki67 staining. Dual staining confirmed that proliferating cells labeled with proSP-B, demonstrating that ventilation induces proliferation of alveolar type II cells. Ventilation did not increase apoptosis in alveolar type II cells, as measured by TUNEL staining. Ventilation at low tidal volumes leads to a mild inflammatory response and alveolar epithelial cell proliferation. PMID:20653468

  7. Phenomenological approaches to collective behavior in epithelial cell migration.

    PubMed

    Zorn, Matthias L; Marel, Anna-Kristina; Segerer, Felix J; Rädler, Joachim O

    2015-11-01

    Collective cell migration in epithelial tissues resembles fluid-like behavior in time-lapse recordings. In the last years, hydrodynamic velocity fields in living matter have been studied intensely. The emergent properties were remarkably similar to phenomena known from active soft matter systems. Here, we review migration experiments of large cellular ensembles as well as of mesoscopic cohorts in micro-structured environments. Concepts such as diffusion, velocity correlations, swirl strength and polarization are metrics to quantify the cellular dynamics both in experiments as well as in computational simulations. We discuss challenges relating collective migration to single cell and oligocellular behavior as well as linking the phenotypic parameters to the underlying cytoskeleton dynamics and signaling networks. This article is part of a Special Issue entitled: Mechanobiology. PMID:26028592

  8. 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...

  9. Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells

    PubMed Central

    Karuri, Nancy W.; Liliensiek, Sara; Teixeira, Ana I.; Abrams, George; Campbell, Sean; Nealey, Paul F.; Murphy, Christopher J.

    2006-01-01

    Summary The basement membrane possesses a rich 3-dimensional nanoscale topography that provides a physical stimulus, which may modulate cell-substratum adhesion. We have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. SV40 human corneal epithelial cells were challenged by well-defined fluid shear, and cell detachment was monitored. We created silicon substrata with uniform grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. F-actin labeling of cells that had been incubated for 24 hours revealed that the percentage of aligned and elongated cells on the patterned surfaces was the same regardless of pitch dimension. In contrast, at the highest fluid shear, a biphasic trend in cell adhesion was observed with cells being most adherent to the smaller features. The 400 nm pitch had the highest percentage of adherent cells at the end of the adhesion assay. The effect of substratum topography was lost for the largest features evaluated, the 4000 nm pitch. Qualitative and quantitative analyses of the cells during and after flow indicated that the aligned and elongated cells on the 400 nm pitch were more tightly adhered compared to aligned cells on the larger patterns. Selected experiments with primary cultured human corneal epithelial cells produced similar results to the SV40 human corneal epithelial cells. These findings have relevance to interpretation of cell-biomaterial interactions in tissue engineering and prosthetic design. PMID:15226393

  10. Single Dose Pharmacokinetics of Oral Tenofovir in Plasma, Peripheral Blood Mononuclear Cells, Colonic Tissue, and Vaginal Tissue

    PubMed Central

    Louissaint, Nicolette A.; Cao, Ying-Jun; Skipper, Paul L.; Liberman, Rosa G.; Tannenbaum, Steven R.; Nimmagadda, Sridhar; Anderson, Jean R.; Everts, Stephanie; Bakshi, Rahul; Fuchs, Edward J.

    2013-01-01

    Abstract HIV seroconversion outcomes in preexposure prophylaxis (PrEP) trials of oral tenofovir (TFV)-containing regimens are highly sensitive to drug concentration, yet less-than-daily dosing regimens are under study. Description of TFV and its active moiety, TFV diphosphate (TFV-DP), in blood, vaginal tissue, and colon tissue may guide the design and interpretation of PrEP clinical trials. Six healthy women were administered a single oral dose of 300 mg tenofovir disoproxil fumarate (TDF) and 4.3 mg (12.31 MBq, 333 μCi) 14C-TDF slurry. Blood was collected every 4 h for the first 24 h, then at 4, 8, 11, and 15 days postdosing. Colonic and vaginal samples (tissue, total and CD4+ cells, luminal fluid and cells) were collected 1, 8 and 15 days postdose. Samples were analyzed for TFV and TFV-DP. Plasma TFV demonstrated triphasic decay with terminal elimination half-life median [interquartile range (IQR)] 69 h (58–77). Peripheral blood mononuclear cell (PBMC) TFV-DP demonstrated biphasic peaks (median 12 h and 96 h) followed by a terminal 48 h (38–76) half-life; Cmax was 20 fmol/million cells (2–63). One day postdose, the TFV-DP paired colon:vaginal tissue concentration ratio was 1 or greater in all subjects' tissue homogenates, median 124 (range 1–281), but was not sustained. The ratio was lower and more variable in cells extracted from tissue. Among all sample types, TFV and TFV-DP half-life ranged from 23 to 139 h. PBMC TFV-DP rose slowly in the hours after dosing indicating that success with exposure-driven dosing regimens may be sensitive to timing of the dose prior to exposure. Colonic tissue homogenate TFV-DP concentrations were greater than in vaginal homogenate at 24 h, but not in cells extracted from tissue. These and the other pharmacokinetic findings will guide the interpretation and design of future PrEP trials. PMID:23600365

  11. Phase I Study of Intravenous Triapine (IND # 68338) in Combination With Pelvic Radiation Therapy With or Without Weekly Intravenous Cisplatin Chemotherapy for Locally Advanced Cervical, Vaginal, or Pelvic Gynecologic Malignancies

    ClinicalTrials.gov

    2013-01-10

    Recurrent Cervical Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Vaginal Cancer; Recurrent Vulvar Cancer; Stage III Vaginal Cancer; Stage IIIA Cervical Cancer; Stage IIIA Ovarian Epithelial Cancer; Stage IIIA Vulvar Cancer; Stage IIIB Cervical Cancer; Stage IIIB Ovarian Epithelial Cancer; Stage IIIB Vulvar Cancer; Stage IIIC Ovarian Epithelial Cancer; Stage IIIC Vulvar Cancer; Stage IV Ovarian Epithelial Cancer; Stage IVA Cervical Cancer; Stage IVA Vaginal Cancer; Stage IVB Cervical Cancer; Stage IVB Vaginal Cancer

  12. Establishment and Characterization of a Buffalo (Bubalus bubalis) Mammary Epithelial Cell Line

    PubMed Central

    Anand, Vijay; Dogra, Nilambra; Singh, Surender; Kumar, Sudarshan N.; Jena, Manoj K.; Malakar, Dhruba; Dang, Ajay K.; Mishra, Bishnu P.; Mukhopadhyay, Tapas K.; Kaushik, Jai K.; Mohanty, Ashok K.

    2012-01-01

    Background The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions. Methodology Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot. Principal Findings The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of ?-casein, ?-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n?=?50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence. Conclusions We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions. PMID:22792341

  13. Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor.

    PubMed

    Yang, Jibing; Velikoff, Miranda; Canalis, Ernesto; Horowitz, Jeffrey C; Kim, Kevin K

    2014-04-15

    Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-?, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-?-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis. PMID:24508728

  14. Increased programmed death-ligand-1 expression in human gastric epithelial cells in Helicobacter pylori infection

    PubMed Central

    Wu, Y-Y; Lin, C-W; Cheng, K-S; Lin, C; Wang, Y-M; Lin, I-T; Chou, Y-H; Hsu, P-N

    2010-01-01

    B7-H1 [programmed death-ligand-1 (PD-L1)] is a B7-family member that binds to programmed death-1 (PD-1). Recently, deficiency of PD-L1 has been demonstrated to result in accelerated gastric epithelial cell damage in gastritis, and PD-L1 is suggested to play a critical role in regulating T cell homeostasis. Here, we aimed to gain more insight into gastric PD-L1 expression, regulation and function during Helicobacter pylori infection. PD-L1 expression in human gastric epithelial cells was analysed using Western blotting, quantitative polymerase chain reaction and fluorescence activated cell sorter analysis. Furthermore, co-culture experiments of human gastric epithelial cells with primary human T cells or Jurkat T cells were conducted. PD-L1 expression in primary human gastric epithelial cells was strongly enhanced by H. pylori infection and activated T cells, and augmented markedly by further stimulation with interferon-? or tumour necrosis factor-?. Moreover, PD-L1 expression in gastric epithelial cells significantly induced apoptosis of T cells. Our results indicate that a novel bidirectional interaction between human gastric epithelial cells and lymphocytes modulates PD-L1 expression in human gastric epithelial cells, contributing to the unique immunological properties of the stomach. PMID:20646001

  15. Cyclin D1 affects epithelialmesenchymal transition in epithelial ovarian cancer stem cell-like cells

    PubMed Central

    Jiao, Jie; Huang, Lu; Ye, Feng; Shi, MinFeng; Cheng, XiaoDong; Wang, XinYu; Hu, DongXiao; Xie, Xing; Lu, WeiGuo

    2013-01-01

    Background The association of cancer stem cells with epithelialmesenchymal transition (EMT) is receiving attention. We found in our previous study that EMT existed from CD24? phenotype cells to their differentiated cells. It was shown that cyclin D1 functioned in sustaining self-renewal independent of CDK4/CDK6 activation, but its effect on the EMT mechanism in ovarian cancer stem cells is unclear. Methods The anchorage-independent spheroids from ovarian adenocarcinoma cell line 3AO were formed in a serum-free medium. CD24? and CD24+ cells were isolated by fluorescence-activated cell sorting. Cell morphology, viability, apoptosis, and migratory ability were observed. Stem-related molecule Bmi-1, Oct-4 and EMT-related marker E-cadherin, and vimentin expressions were analyzed. Cyclin D1 expression in CD24? phenotype enriched spheroids was knocked down with small interfering RNA, and its effects on cell proliferation, apoptosis, migration ability, and EMT-related phenotype after transfection were observed. Results In our study, CD24? cells presented stronger proliferative, anti-apoptosis capacity, and migratory ability, than CD24+ cells or parental cells. CD24? cells grew with a scattered spindle-shape within 3 days of culture and transformed into a cobblestone-like shape, identical to CD24+ cells or parental cells at 7 days of culture. CD24? cells or spheroids highly expressed cyclin D1, Bmi-1, and vimentin, and seldom expressed E-cadherin, while CD24+ or parental cells showed the opposite expression. Furthermore, cyclin D1-targeted small interfering RNA resulted in decreased vimentin expression in spheroids. Transfected cells also exhibited an obvious decrease in cell viability and migration, but an increase in cell apoptosis. Conclusion Cancer stem cell-like cells possess mesenchymal characteristics and EMT ability, and cyclin D1 involves in EMT mechanism, suggesting that EMT of cancer stem cell-like cells may play a key role in invasion and metastasis of ovarian cancer. PMID:23836980

  16. CAR regulates epithelial cell junction stability through control of E-cadherin trafficking

    PubMed Central

    Morton, Penny E.; Hicks, Alexander; Nastos, Theodoros; Santis, George; Parsons, Maddy

    2013-01-01

    CAR (Coxsackie and Adenovirus Receptor) is the primary docking receptor for typeB coxsackie viruses and subgroup C adenoviruses. CAR is a member of the JAM family of adhesion receptors and is located to both tight and adherens junctions between epithelial cells where it can assemble adhesive contacts through homodimerisation in trans. However, the role of CAR in controlling epithelial junction dynamics remains poorly understood. Here we demonstrate that levels of CAR in human epithelial cells play a key role in determining epithelial cell adhesion through control of E-cadherin stability at cell-cell junctions. Mechanistically, we show that CAR is phosphorylated within the C-terminus by PKCδ and that this in turn controls Src-dependent endocytosis of E-cadherin at cell junctions. This data demonstrates a novel role for CAR in regulating epithelial homeostasis. PMID:24096322

  17. Integrins regulate epithelial cell differentiation by modulating Notch activity

    PubMed Central

    Gmez-Lamarca, M. Jess; Cobreros-Reguera, Laura; Ibez-Jimnez, Beatriz; Palacios, Isabel M.; Martn-Bermudo, Mara D.

    2014-01-01

    ABSTRACT Coordinating exit from the cell cycle with differentiation is crucial for proper development and tissue homeostasis. Failure to do so can lead to aberrant organogenesis and tumorigenesis. However, little is known about the developmental signals that regulate the switch from cell cycle exit to differentiation. Signals downstream of two key developmental pathways, Notch and SalvadorWartsHippo (SWH), and signals downstream of myosin activity regulate this switch during the development of the follicle cell epithelium of the Drosophila ovary. Here, we have identified a fourth player, the integrin signaling pathway. Elimination of integrin function blocks the mitosis-to-endocycle switch and differentiation in posterior follicle cells (PFCs), by regulation of the cyclin-dependent kinase inhibitor (CKI) dacapo. In addition, integrin-mutant PFCs show defective Notch signaling and endocytosis. Furthermore, integrins act in PFCs by modulating the activity of the Notch pathway, as reducing the amount of Hairless, the major antagonist of Notch, or misexpressing Notch intracellular domain rescues the cell cycle and differentiation defects. Taken together, our findings reveal a direct involvement of integrin signaling on the spatial and temporal regulation of epithelial cell differentiation during development. PMID:25179603

  18. Ionizing radiation induces heritable disruption of epithelial cell interactions

    NASA Technical Reports Server (NTRS)

    Park, Catherine C.; Henshall-Powell, Rhonda L.; Erickson, Anna C.; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Chatterjee, A. (Principal Investigator)

    2003-01-01

    Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

  19. Role of the microtubule-targeting drug vinflunine on cell-cell adhesions in bladder epithelial tumour cells

    PubMed Central

    2014-01-01

    Background Vinflunine (VFL) is a microtubule-targeting drug that suppresses microtubule dynamics, showing anti-metastatic properties both in vitro and in living cancer cells. An increasing body of evidence underlines the influence of the microtubules dynamics on the cadherin-dependent cell-cell adhesions. E-cadherin is a marker of epithelial-to-mesenchymal transition (EMT) and a tumour suppressor; its reduced levels in carcinoma are associated with poor prognosis. In this report, we investigate the role of VFL on cell-cell adhesions in bladder epithelial tumour cells. Methods Human bladder epithelial tumour cell lines HT1376, 5637, SW780, T24 and UMUC3 were used to analyse cadherin-dependent cell-cell adhesions under VFL treatment. VFL effect on growth inhibition was measured by using a MTT colorimetric cell viability assay. Western blot, immunofluorescence and transmission electron microscopy analyses were performed to assess the roles of VFL effect on cell-cell adhesions, epithelial-to-mesenchymal markers and apoptosis. The role of the proteasome in controlling cell-cell adhesion was studied using the proteasome inhibitor MG132. Results We show that VFL induces cell death in bladder cancer cells and activates epithelial differentiation of the remaining living cells, leading to an increase of E-cadherin-dependent cell-cell adhesion and a reduction of mesenchymal markers, such as N-cadherin or vimentin. Moreover, while E-cadherin is increased, the levels of Hakai, an E3 ubiquitin-ligase for E-cadherin, were significantly reduced in presence of VFL. In 5637, this reduction on Hakai expression was blocked by MG132 proteasome inhibitor, indicating that the proteasome pathway could be one of the molecular mechanisms involved in its degradation. Conclusions Our findings underscore a critical function for VFL in cell-cell adhesions of epithelial bladder tumour cells, suggesting a novel molecular mechanism by which VFL may impact upon EMT and metastasis. PMID:25012153

  20. 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

  1. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

    PubMed Central

    Playford, Martin P.; Vadali, Kavita; Cai, Xinming; Burridge, Keith; Schaller, Michael D.

    2008-01-01

    Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho. PMID:18773890

  2. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

    SciTech Connect

    Playford, Martin P.; Vadali, Kavita; Cai Xinming; Burridge, Keith; Schaller, Michael D.

    2008-10-15

    Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.

  3. A novel type of cell-cell cooperation between epithelial cells.

    PubMed

    Contreras, R G; Lázaro, A; Bolivar, J J; Flores-Maldonado, C; Sánchez, S H; González-Mariscal, L; García-Villegas, M R; Valdés, J; Cereijido, M

    1995-06-01

    Ma104 cells (renal, epithelial) have a peculiar way of resisting ouabain: their Na+,K(+)-pumps bind the drug with high affinity, cellular K+ is lost and cell division arrested, but cells do not detach as most cell types do. Then, if up to 4 days later the drug is removed, Ma104 cells recover K+ and resume proliferation (Contreras et al., 1994). In the present work, we investigate whether Ma104 cells are able to protect ouabain-sensitive MDCK cells in co-culture. The main finding is that they do, but in this case protection is not elicited by the usual mechanism of maintaining the K+ content of neighboring cells through cell-cell communications. Ma104 cells treated with ouabain simply remain attached to the substrate and to their MDCK neighbors, and both cells lose K+. This attachment includes tight junctions, because the transepithelial electrical resistance of the monolayers is not abolished by ouabain. Although the beta-subunit of the Na+,K(+)-ATPase is known to possess molecular characteristics of cell-cell attachment molecules, attachment between Ma104-MDCK cells does not seem to be mediated by this enzyme, as immunofluorescence analysis reveals that Na+,K(+)-ATPase is only inserted in the plasma membrane facing a neighboring cell of the same type. PMID:7563031

  4. Topical KGF treatment as a therapeutic strategy for vaginal atrophy in a model of ovariectomized mice

    PubMed Central

    Ceccarelli, Simona; D'Amici, Sirio; Vescarelli, Enrica; Coluccio, Paolo; Matricardi, Pietro; di Gioia, Cira; Benedetti Panici, Pierluigi; Romano, Ferdinando; Frati, Luigi; Angeloni, Antonio; Marchese, Cinzia

    2014-01-01

    One of the most frequent complaints for post-menopausal women is vaginal atrophy, because of reduction in circulating oestrogens. Treatments based on local oestrogen administration have been questioned as topic oestrogens can reach the bloodstream, thus leading to consider their safety as controversial, especially for patients with a history of breast or endometrial cancers. Recently, growth factors have been shown to interact with the oestrogen pathway, but the mechanisms still need to be fully clarified. In this study, we investigated the effect of keratinocyte growth factor (KGF), a known mitogen for epithelial cells, on human vaginal mucosa cells, and its potential crosstalk with oestrogen pathways. We also tested the in vivo efficacy of KGF local administration on vaginal atrophy in a murine model. We demonstrated that KGF is able to induce proliferation of vaginal mucosa, and we gained insight on its mechanism of action by highlighting its contribution to switch ERα signalling towards non-genomic pathway. Moreover, we demonstrated that KGF restores vaginal trophism in vivo similarly to intravaginal oestrogenic preparations, without systemic effects. Therefore, we suggest a possible alternative therapy for vaginal atrophy devoid of the risks related to oestrogen-based treatments, and a patent (no. RM2012A000404) has been applied for this study. PMID:25088572

  5. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency.

    PubMed

    Utheim, Tor Paaske; Utheim, Øygunn Aass; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-01-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells. PMID:26938569

  6. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells.

    PubMed

    Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A; Costa, Max

    2016-02-15

    The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. PMID:26780400

  7. Sphere formation permits Oct4 reprogramming of ciliary body epithelial cells into induced pluripotent stem cells.

    PubMed

    Ni, Aiguo; Wu, Ming Jing; Chavala, Sai H

    2014-12-15

    Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by defined sets of transcription factors. We previously described reprogramming of monolayer-cultured adult mouse ciliary body epithelial (CE) cells by Oct4 and Klf4, but not with Oct4 alone. In this study, we report that Oct4 alone is sufficient to reprogram CE cells to iPS cells through sphere formation. Furthermore, we demonstrate that sphere formation induces a partial reprogramming state characterized by expression of retinal progenitor markers, upregulation of reprogramming transcription factors, such as Sall4 and Nanog, demethylation in the promoter regions of pluripotency associated genes, and mesenchymal to epithelial transition. The Oct4-iPS cells maintained normal karyotypes, expressed markers for pluripotent stem cells, and were capable of differentiating into derivatives of all three embryonic germ layers in vivo and in vitro. These findings suggest that sphere formation may render somatic cells more susceptible to reprogramming. PMID:25027059

  8. Deletion of Lkb1 in Renal Tubular Epithelial Cells Leads to CKD by Altering Metabolism.

    PubMed

    Han, Seung Hyeok; Malaga-Dieguez, Laura; Chinga, Frank; Kang, Hyun Mi; Tao, Jianling; Reidy, Kimberly; Susztak, Katalin

    2016-02-01

    Renal tubule epithelial cells are high-energy demanding polarized epithelial cells. Liver kinase B1 (LKB1) is a key regulator of polarity, proliferation, and cell metabolism in epithelial cells, but the function of LKB1 in the kidney is unclear. Our unbiased gene expression studies of human control and CKD kidney samples identified lower expression of LKB1 and regulatory proteins in CKD. Mice with distal tubule epithelial-specific Lkb1 deletion (Ksp-Cre/Lkb1(flox/flox)) exhibited progressive kidney disease characterized by flattened dedifferentiated tubule epithelial cells, interstitial matrix accumulation, and dilated cystic-appearing tubules. Expression of epithelial polarity markers ?-catenin and E-cadherin was not altered even at later stages. However, expression levels of key regulators of metabolism, AMP-activated protein kinase (Ampk), peroxisome proliferative activated receptor gamma coactivator 1-? (Ppargc1a), and Ppara, were significantly lower than those in controls and correlated with fibrosis development. Loss of Lkb1 in cultured epithelial cells resulted in energy depletion, apoptosis, less fatty acid oxidation and glycolysis, and a profibrotic phenotype. Treatment of Lkb1-deficient cells with an AMP-activated protein kinase (AMPK) agonist (A769662) or a peroxisome proliferative activated receptor alpha agonist (fenofibrate) restored the fatty oxidation defect and reduced apoptosis. In conclusion, we show that loss of LKB1 in renal tubular epithelial cells has an important role in kidney disease development by influencing intracellular metabolism. PMID:26054542

  9. Diet Does Not Affect Putative Mammary Epithelial Stem Cells in Pre-weaned Holstein Heifers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Overfeeding prepubertal heifers can impair mammary epithelial growth and development, processes that depend on stem cells. In this study we evaluated effects of diet composition on putative bovine mammary epithelial stem cell populations using a 5-bromo-2-deoxyrudine (BrdU; a thymidine analog) label...

  10. ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCTED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS

    EPA Science Inventory

    ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS.
    OBJECTIVE: We have shown that functional gap junction communication as measured by Lucifer yellow dye transfer (DT) in Clone-9 rat liver epithelial cells, c...

  11. Roles of Wnt/{beta}-catenin signaling in epithelial differentiation of mesenchymal stem cells

    SciTech Connect

    Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Jiangsu Key Laboratory of Molecular Medicine, Nanjing 210093 ; Li, Yan; Qin, Jizheng; Jiangsu Key Laboratory of Molecular Medicine, Nanjing 210093 ; Han, Xiaodong; Jiangsu Key Laboratory of Molecular Medicine, Nanjing 210093

    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.

  12. Regulation of epithelial cell organization by tuning cell-substrate adhesion.

    PubMed

    Ravasio, Andrea; Le, Anh Phuong; Saw, Thuan Beng; Tarle, Victoria; Ong, Hui Ting; Bertocchi, Cristina; Mge, Ren-Marc; Lim, Chwee Teck; Gov, Nir S; Ladoux, Benoit

    2015-10-01

    Collective migration of cells is of fundamental importance for a number of biological functions such as tissue development and regeneration, wound healing and cancer metastasis. The movement of cell groups consisting of multiple cells connected by cell-cell junctions depends on both extracellular and intercellular contacts. Epithelial cell assemblies are thus regulated by a cross-talk between cell-substrate and cell-cell interactions. Here, we investigated the onset of collective migration in groups of cells as they expand from a few cells into large colonies as a function of extracellular matrix (ECM) protein coating. By varying the amount of ECM presented to the cells, we observe that the mode of colony expansion, as well as their overall geometry, is strongly dependent on substrate adhesiveness. On high ECM protein coated surfaces, cells at the edges of the colonies are well spread exhibiting large outward-pointing protrusive activity, whereas cellular colonies display more circular and convex shapes on less adhesive surfaces. Actin structures at the edge of the colonies also show different organizations with the formation of lamellipodial structures on highly adhesive surfaces and a pluricellular actin cable on less adhesive ones. The analysis of traction forces and cell velocities within the cellular assemblies confirm these results. By increasing ECM protein density, cells exert higher traction forces together with a higher outward motility at the edges. Furthermore, tuning cell-cell adhesion of epithelial cells modified the mode of expansion of the colonies. Finally, we used a recently developed computational model to recapitulate the emergent experimental behaviors of expanding cell colonies and extract that the main effect of the different cell-substrate interactions is on the ability of edge cells to form outward lamellipodia-driven motility. Overall, our data suggest that switching behaviors of epithelial cell assemblies result in a tug-of-war between friction forces at the cell-substrate interface and cell-cell interactions. PMID:26402903

  13. Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin.

    PubMed Central

    Yoshimura, K; Nakamura, H; Trapnell, B C; Chu, C S; Dalemans, W; Pavirani, A; Lecocq, J P; Crystal, R G

    1991-01-01

    Consistent with the fact that the clinical disorder cystic fibrosis (CF) is manifested on epithelial surfaces, active transcription of the CF transmembrane conductance regulator (CFTR) gene and CFTR mRNA transcripts are detectable in a variety of epithelial cells, suggesting CFTR gene expression might be epithelial cell-specific. However, analysis of the CFTR gene promoter suggests it is a housekeeping gene, implying more widespread expression than only in epithelial cells. To evaluate the latter hypothesis, various human cells of non-epithelial origin, including lung fibroblasts, U-937 histiocytic lymphoma cells, K-562 erythroleukemia cells, HL-60 promyelocytic leukemia cells as well as freshly isolated blood lymphocytes, neutrophils, monocytes, and alveolar macrophages were examined for CFTR gene expression. Although Northern analysis failed to show CFTR mRNA transcripts in these cells, amplification of mRNA (after conversion to cDNA) by polymerase chain reaction combined with Southern analysis demonstrated the presence of CFTR mRNA transcripts at low levels in all cells evaluated except HL-60 cells. Comparative quantitative analysis showed fibroblasts contained 200-400 fold less CFTR mRNA transcripts than the T84 and HT-29 colon carcinoma epithelial cell lines, but had similar levels of CFTR transcripts to those of other epithelial cell lines. Nuclear transcription run-on analyses demonstrated very low level CFTR gene transcription in fibroblasts and U-937 cells, similar to that of other epithelial cells, but lower than the T84 and HT-29 colon carcinoma cell lines. Interestingly, while chromatin DNA of fibroblasts had no DNase I hypersensitivity sites in the 5' flanking region of the CFTR gene, HT-29 chromatin DNA exhibited four DNase I accessible sites in the same region, suggesting that these sites may be related to more active transcription of the CFTR gene in the intestinal epithelial cells than in fibroblasts. Images PMID:1717947

  14. Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules.

    PubMed

    Marrs, J A; Andersson-Fisone, C; Jeong, M C; Cohen-Gould, L; Zurzolo, C; Nabi, I R; Rodriguez-Boulan, E; Nelson, W J

    1995-04-01

    A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E-cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types. PMID:7536748

  15. ?-Amylase in Vaginal Fluid: Association With Conditions Favorable to Dominance of Lactobacillus.

    PubMed

    Nasioudis, Dimitrios; Beghini, Joziani; Bongiovanni, Ann Marie; Giraldo, Paulo C; Linhares, Iara M; Witkin, Steven S

    2015-11-01

    Vaginal glycogen is degraded by host ?-amylase and then converted to lactic acid by Lactobacilli. This maintains the vaginal pH at ?4.5 and prevents growth of other bacteria. Therefore, host ?-amylase activity may promote dominance of Lactobacilli. We evaluated whether the ?-amylase level in vaginal fluid is altered in women with bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) and whether its concentration was associated with levels of lactic acid isomers and host mediators. Vaginal fluid was obtained from 43 women with BV, 50 women with VVC, and 62 women with no vulvovaginal disorders. Vaginal fluid concentrations of ?-amylase, secretory leukocyte protease inhibitor (SLPI), hyaluronan, hyaluronidase-1, ?-defensin, and elafin were measured by enzyme-linked immunosorbent assay (ELISA). Vaginal concentrations of neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP) 8, and d- and l-lactic acid levels in these patients were previously reported. The median vaginal fluid ?-amylase level was 1.83 mU/mL in control women, 1.45 mU/mL in women with VVC, and 1.07 mU/mL in women with BV. Vaginal levels of ?-amylase were correlated with d-lactic acid (P = .003) but not with l-lactic acid (P > .05) and with SLPI (P < .001), hyaluronidase-1 (P < .001), NGAL (P = .001), and MMP-8 (P = .005). The exfoliation of glycogen-rich epithelial cells into the vaginal lumen by hyaluronidase-1 and MMP-8 may increase glycogen availability and promote ?-amylase activity. The subsequent enhanced availability of glycogen breakdown products would favor proliferation of Lactobacilli, the primary producers of d-lactic acid in the vagina. Concomitant production of NGAL and SLPI would retard growth of BV-related bacteria. PMID:25878210

  16. Lactobacillus crispatus L1: high cell density cultivation and exopolysaccharide structure characterization to highlight potentially beneficial effects against vaginal pathogens

    PubMed Central

    2014-01-01

    Background Vaginal lactic acid bacteria defend the host against pathogens through a combination of competitive exclusion, competition for nutrients, production of antimicrobial substances and through the activation of the immune system. A new human isolate named Lactobacillus crispatus L1 was characterized in this work, and a preliminary evaluation of its probiotic potential is described together with a process to obtain a high productivity of viable biomass. Results In a simulated digestion process 1.8⋅1010 cells∙ml−1 survived the gastric environment with 80% viability, without being affected by small intestine juices. Experiments on six different C sources were performed to analyze growth and organic acids production and, glucose, provided the best performances. A microfiltration strategy was exploited to improve the cellular yield in 2 L-fermentation processes, reaching 27 g · l−1 of dry biomass. Moreover, L. crispatus L1 demonstrated a greater stability to high concentrations of lactic acid, compared to other lactobacilli. The specific L. crispatus L1 exopolysaccharide was purified from the fermentation broth and characterized by NMR showing structural features and similarity to exopolysaccharides produced by pathogenic strains. Live L. crispatus L1 cells strongly reduced adhesion of a yeast pathogenic strain, Candida albicans in particular, in adherence assays. Interestingly a higher expression of the human defensin HBD-2 was also observed in vaginal cells treated with the purified exopolysaccharide, indicating a possible correlation with C. albicans growth inhibition. Conclusions The paper describes the evaluation of L. crispatus L1 as potential vaginal probiotic and the fermentation processes to obtain high concentrations of viable cells. PMID:24884965

  17. Spatiotemporally Regulated Ablation of Klf4 in Adult Mouse Corneal Epithelial Cells Results in Altered Epithelial Cell Identity and Disrupted Homeostasis

    PubMed Central

    Delp, Emili E.; Swamynathan, Sudha; Kao, Winston W.; Swamynathan, Shivalingappa K.

    2015-01-01

    Purpose. In previous studies, conditional disruption of Klf4 in the developing mouse ocular surface from embryonic day 10 resulted in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells, revealing the importance of Klf4 in ocular surface maturation. Here, we use spatiotemporally regulated ablation of Klf4 to investigate its functions in maintenance of adult corneal epithelial homeostasis. Methods. Expression of Cre was induced in ternary transgenic (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mouse corneal epithelium by doxycycline administered through intraperitoneal injections and drinking water, to generate corneal epithelium–specific deletion of Klf4 (Klf4Δ/ΔCE). Corneal epithelial barrier function was tested by fluorescein staining. Expression of selected Klf4-target genes was determined by quantitative PCR (QPCR), immunoblotting, and immunofluorescent staining. Results. Klf4 was efficiently ablated within 5 days of doxycycline administration in adult Klf4Δ/ΔCE corneal epithelium. The Klf4Δ/ΔCE corneal epithelial barrier function was disrupted, and the basal cells were swollen and rounded after 15 days of doxycycline treatment. Increased numbers of cell layers and Ki67-positive proliferating cells suggested deregulated Klf4Δ/ΔCE corneal epithelial homeostasis. Expression of tight junction proteins ZO-1 and occludin, desmosomal Dsg and Dsp, basement membrane laminin-332, and corneal epithelial–specific keratin-12 was decreased, while that of matrix metalloproteinase Mmp9 and noncorneal keratin-17 increased, suggesting altered Klf4Δ/ΔCE corneal epithelial cell identity. Conclusions. Ablation of Klf4 in the adult mouse corneas resulted in the absence of characteristic corneal epithelial cell differentiation, disrupted barrier function, and squamous metaplasia, revealing that Klf4 is essential for maintenance of the adult corneal epithelial cell identity and homeostasis. PMID:26047041

  18. Endothelial induced EMT in breast epithelial cells with stem cell properties.

    PubMed

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

    2011-01-01

    Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer. PMID:21915264

  19. Mechanical stimulation of epithelial cells using polypyrrole microactuators.

    PubMed

    Svennersten, Karl; Berggren, Magnus; Richter-Dahlfors, Agneta; Jager, Edwin W H

    2011-10-01

    The importance of mechanotransduction for physiological systems is becoming increasingly recognized. The effect of mechanical stimulation is well studied in organs and tissues, for instance by using flexible tissue culture substrates that can be stretched by external means. However, on the cellular and subcellular level, dedicated technology to apply appropriate mechanical stimuli is limited. Here we report an organic electronic microactuator chip for mechanical stimulation of single cells. These chips are manufactured on silicon wafers using traditional microfabrication and photolithography techniques. The active unit of the chip consists of the electroactive polymer polypyrrole that expands upon the application of a low potential. The fact that polypyrrole can be activated in physiological electrolytes makes it well suited as the active material in a microactuator chip for biomedical applications. Renal epithelial cells, which are responsive to mechanical stimuli and relevant from a physiological perspective, are cultured on top of the microactuator chip. The cells exhibit good adhesion and spread along the surface of the chip. After culturing, individual cells are mechanically stimulated by electrical addressing of the microactuator chip and the response to this stimulation is monitored as an increase in intracellular Ca(2+). This Ca(2+) response is caused by an autocrine ATP signalling pathway associated with mechanical stimulation of the cells. In conclusion, the present work demonstrates a microactuator chip based on an organic conjugated polymer, for mechanical stimulation of biological systems at the cellular and sub-cellular level. PMID:21842071

  20. Radiation-induced chromosomal instability in human mammary epithelial cells

    NASA Technical Reports Server (NTRS)

    Durante, M.; Grossi, G. F.; Yang, T. C.

    1996-01-01

    Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

  1. 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/.

  2. Radiation-induced chromosomal instability in human mammary epithelial cells

    NASA Astrophysics Data System (ADS)

    Durante, M.; Grossi, G. F.; Yang, T. C.

    Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

  3. ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis

    PubMed Central

    Guo, Feiye; Ding, Ying; Caberoy, Nora; Alvarado, Gabriela; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis of shed photoreceptor outer segments (POSs) by retinal pigment epithelial (RPE) cells is critical to retinal homeostasis and shares many conserved signaling pathways with other phagocytes, including extrinsic regulations. Phagocytotic ligands are the key to cargo recognition, engulfment initiation, and activity regulation. In this study, we identified intracellular protein ATP-binding cassette subfamily F member 1 (ABCF1) as a novel RPE phagocytotic ligand by a new approach of functional screening. ABCF1 was independently verified to extrinsically promote phagocytosis of shed POSs by D407 RPE cells. This finding was further corroborated with primary RPE cells and RPE explants. Internalized POS vesicles were colocalized with a phagosome marker, suggesting that ABCF1-mediated engulfment is through a phagocytic pathway. ABCF1 was released from apoptotic cells and selectively bound to shed POS vesicles and apoptotic cells, possibly via externalized phosphatidylserine. ABCF1 is predominantly expressed in POSs and colocalized with the POS marker rhodopsin, providing geographical convenience for regulation of RPE phagocytosis. Collectively these results suggest that ABCF1 is released from and binds to shed POSs in an autocrine manner to facilitate RPE phagocytosis through a conserved pathway. Furthermore, the new approach is broadly applicable to many other phagocytes and will enable systematic elucidation of their ligands to understand extrinsic regulation and cargo recognition. PMID:25904329

  4. Disruption of the keratin filament network during epithelial cell division.

    PubMed Central

    Lane, E B; Goodman, S L; Trejdosiewicz, L K

    1982-01-01

    The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6202508

  5. The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity

    PubMed Central

    Ayala, Inmaculada; Colanzi, Antonino; Lapazio, Lucia; Corda, Daniela; Soriani, Marco; Pizza, Mariagrazia; Rossi Paccani, Silvia

    2015-01-01

    Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis. PMID:25996923

  6. The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

    PubMed

    Valeri, Maria; Zurli, Vanessa; Ayala, Inmaculada; Colanzi, Antonino; Lapazio, Lucia; Corda, Daniela; Soriani, Marco; Pizza, Mariagrazia; Rossi Paccani, Silvia

    2015-01-01

    Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis. PMID:25996923

  7. A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

    PubMed Central

    Choudhary, Parul; Dodsworth, Benjamin Thomas; Sidders, Ben; Gutteridge, Alex; Michaelides, Christos; Duckworth, Joshua Kane; Whiting, Paul John; Benn, Caroline Louise

    2015-01-01

    The integrity of the epithelium is maintained by a complex but regulated interplay of processes that allow conversion of a proliferative state into a stably differentiated state. In this study, using human embryonic stem cell (hESC) derived Retinal Pigment Epithelium (RPE) cells as a model; we have investigated the molecular mechanisms that affect attainment of the epithelial phenotype. We demonstrate that RPE undergo a Mesenchymal–Epithelial Transition in culture before acquiring an epithelial phenotype in a FOXM1 dependent manner. We show that FOXM1 directly regulates proliferation of RPE through transcriptional control of cell cycle associated genes. Additionally, FOXM1 modulates expression of the signaling ligands BMP7 and Wnt5B which act reciprocally to enable epithelialization. This data uncovers a novel effect of FOXM1 dependent activities in contributing towards epithelial fate acquisition and furthers our understanding of the molecular regulators of a cell type that is currently being evaluated as a cell therapy. PMID:26121260

  8. Primary cilia and aberrant cell signaling in epithelial ovarian cancer

    PubMed Central

    2012-01-01

    Background Ovarian cancer is the fourth leading cause of cancer-related deaths among women in Denmark, largely due to the advanced stage at diagnosis in most patients. Approximately 90% of ovarian cancers originate from the single-layered ovarian surface epithelium (OSE). Defects in the primary cilium, a solitary sensory organelle in most cells types including OSE, were recently implicated in tumorigenesis, mainly due to deregulation of ciliary signaling pathways such as Hedgehog (Hh) signaling. However, a possible link between primary cilia and epithelial ovarian cancer has not previously been investigated. Methods The presence of primary cilia was analyzed in sections of fixed human ovarian tissue as well as in cultures of normal human ovarian surface epithelium (OSE) cells and two human OSE-derived cancer cell lines. We also used immunofluorescence microscopy, western blotting, RT-PCR and siRNA to investigate ciliary signaling pathways in these cells. Results We show that ovarian cancer cells display significantly reduced numbers of primary cilia. The reduction in ciliation frequency in these cells was not due to a failure to enter growth arrest, and correlated with persistent centrosomal localization of aurora A kinase (AURA). Further, we demonstrate that ovarian cancer cells have deregulated Hh signaling and platelet-derived growth factor receptor alpha (PDGFR?) expression and that promotion of ciliary formation/stability by AURA siRNA depletion decreases Hh signaling in ovarian cancer cells. Lastly, we show that the tumor suppressor protein and negative regulator of AURA, checkpoint with forkhead-associated and ring finger domains (CHFR), localizes to the centrosome/primary cilium axis. Conclusions Our results suggest that primary cilia play a role in maintaining OSE homeostasis and that the low frequency of primary cilia in cancer OSE cells may result in part from over-expression of AURA, leading to aberrant Hh signaling and ovarian tumorigenesis. PMID:23351307

  9. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

    PubMed Central

    Taylor, AW; Dixit, S; Yu, J

    2015-01-01

    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 addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye. PMID:25905107

  10. Functional characterization of choroid plexus epithelial cells in primary culture.

    PubMed

    Villalobos, A R; Parmelee, J T; Pritchard, J B

    1997-08-01

    The objective of this study was to develop and evaluate a primary culture system for choroid plexus epithelial cells as an in vitro model for studying organic cation transport. Cells were dispersed from choroid plexus of neonatal rats by enzymatic digestion and grew as differentiated monolayers when plated on solid or permeable support. Electron microscopy showed that cultured cells were morphologically similar to intact choroid plexus epithelium, having apical tight junctions between cells, numerous mitochondria, basal nuclei and apical microvilli and cilia. As previously demonstrated for intact choroid plexus, immunocytochemistry showed that Na+,K+-ATPase was localized to the apical membrane, and GLUT-1, the facilitative glucose transporter, was localized to the basolateral membrane of cultured cells. Apical transport of L-proline by cultured cells was mediated by a sodium-dependent, electrogenic process, as in whole tissue. 14C-Tetraethylammonium (TEA), a prototypic organic cation, was accumulated by isolated choroid plexus in a time-dependent manner; uptake was inhibited by tetrapentylammonium (TePA). In cultured cells, apical TEA transport was mediated by a saturable process coupled to cellular metabolism. Unlabeled TEA and other organic cations (TePA, N1-methylnicotinamide and mepiperphenidol) inhibited TEA transport; the organic anion, p-aminohippurate, had no effect. Finally, TePA-sensitive transport of 14C-TEA was stimulated after preloading the cells with unlabeled TEA. Based on the morphological, biochemical and functional properties of these cultured cells, we conclude that this primary culture system should be an excellent in vitro model for experimental characterization of choroid plexus function. PMID:9262381

  11. Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

    PubMed

    Patil, Pratima U; D'Ambrosio, Julia; Inge, Landon J; Mason, Robert W; Rajasekaran, Ayyappan K

    2015-12-01

    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

  12. Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

    PubMed Central

    Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

    2015-01-01

    Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells. PMID:26602832

  13. Survival of Exfoliated Epithelial Cells: A Delicate Balance between Anoikis and Apoptosis

    PubMed Central

    Bertrand, Kaeffer

    2011-01-01

    The recovery of exfoliated cells from biological fluids is a noninvasive technology which is in high demand in the field of translational research. Exfoliated epithelial cells can be isolated from several body fluids (i.e., breast milk, urines, and digestives fluids) as a cellular mixture (senescent, apoptotic, proliferative, or quiescent cells). The most intriguing are quiescent cells which can be used to derive primary cultures indicating that some phenotypes retain clonogenic potentials. Such exfoliated cells are believed to enter rapidly in anoikis after exfoliation. Anoikis can be considered as an autophagic state promoting epithelial cell survival after a timely loss of contact with extracellular matrix and cell neighbors. This paper presents current understanding of exfoliation along with the influence of methodology on the type of gastrointestinal epithelial cells isolated and, finally, speculates on the balance between anoikis and apoptosis to explain the survival of gastrointestinal epithelial cells in the environment. PMID:22131811

  14. Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

    NASA Astrophysics Data System (ADS)

    Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

    2015-11-01

    Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

  15. Human amniotic epithelial cells express specific markers of nerve cells and migrate along the nerve fibers in the corpus callosum?

    PubMed Central

    Wu, Zhiyuan; Hui, Guozhen; Lu, Yi; Liu, Tianjin; Huang, Qin; Guo, Lihe

    2012-01-01

    Human amniotic epithelial cells were isolated from a piece of fresh amnion. Using immunocytochemical methods, we investigated the expression of neuronal phenotypes (microtubule-associated protein-2, glial fibrillary acidic protein and nestin) in human amniotic epithelial cells. The conditioned medium of human amniotic epithelial cells promoted the growth and proliferation of rat glial cells cultured in vitro, and this effect was dose-dependent. Human amniotic epithelial cells were further transplanted into the corpus striatum of healthy adult rats and the grafted cells could integrate with the host and migrate 12 mm along the nerve fibers in corpus callosum. Our experimental findings indicate that human amniotic epithelial cells may be a new kind of seed cells for use in neurograft. PMID:25806057

  16. Intrinsic lens forming potential of mouse lens epithelial versus newt iris pigment epithelial cells in three-dimensional culture.

    PubMed

    Hoffmann, Andrea; Nakamura, Kenta; Tsonis, Panagiotis A

    2014-02-01

    Adult newts (Notophthalmus viridescens) are capable of complete lens regeneration that is mediated through dorsal iris pigment epithelial (IPE) cells transdifferentiation. In contrast, higher vertebrates such as mice demonstrate only limited lens regeneration in the presence of an intact lens capsule with remaining lens epithelial cells. To compare the intrinsic lens regeneration potential of newt IPE versus mouse lens epithelial cells (MLE), we have established a novel culture method that uses cell aggregation before culture in growth factor-reduced Matrigel. Dorsal newt IPE aggregates demonstrated complete lens formation within 1 to 2 weeks of Matrigel culture without basic fibroblast growth factor (bFGF) supplementation, including the establishment of a peripheral cuboidal epithelial cell layer, and the appearance of central lens fibers that were positive for ?A-crystallin. In contrast, the lens-forming potential of MLE cell aggregates cultured in Matrigel was incomplete and resulted in the formation of defined-size lentoids with partial optical transparency. While the peripheral cell layers of MLE aggregates were nucleated, cells in the center of aggregates demonstrated a nonapoptotic nuclear loss over a time period of 3 weeks that was representative of lens fiber formation. Matrigel culture supplementation with bFGF resulted in higher transparent bigger-size MLE aggregates that demonstrated increased appearance of ?B1-crystallin expression. Our study demonstrates that bFGF is not required for induction of newt IPE aggregate-dependent lens formation in Matrigel, while the addition of bFGF seems to be beneficial for the formation of MLE aggregate-derived lens-like structures. In conclusion, the three-dimensional aggregate culture of IPE and MLE in Matrigel allows to a higher extent than older models the indepth study of the intrinsic lens-forming potential and the corresponding identification of lentogenic factors. PMID:23672748

  17. Transport and epithelial secretion of the cardiac glycoside, digoxin, by human intestinal epithelial (Caco-2) cells.

    PubMed Central

    Cavet, M. E.; West, M.; Simmons, N. L.

    1996-01-01

    1. Human intestinal epithelial Caco-2 cells have been used to investigate the transepithelial permeation of the cardiac glycoside, digoxin. 2. Transepithelial basal to apical [3H]-digoxin flux exceeds apical to basal flux, a net secretion of [3H]-digoxin being observed. At 200 microM digoxin, net secretory flux (Jnet) was 10.8 +/- 0.6 nmol cm-2 h-1. Maximal secretory flux (Jmax) of vinblastine was 1.3 +/- 0.1 nmol cm-2 h-1. Cellular uptake of digoxin was different across apical and basal cell boundaries. It was greatest across the basal surface at 1 microM, whereas at 200 microM, apical uptake exceeded basal uptake. 3. Net secretion of [3H]-digoxin was subject to inhibition by digitoxin and bufalin but was not inhibited by ouabain, convallatoxin, and strophanthidin (all 100 microM). Inhibition was due to both a decrease in Jb-a and an increase in Ja-b. Uptake of [3H]-digoxin at the apical surface was increased by digitoxin and bufalin. All cardiac glycosides decreased [3H]-digoxin uptake at the basal cell surface (except for 100 microM digitoxin). 4. The competitive P-glycoprotein inhibitors, verapamil (100 microM), nifedipine (50 microM) and vinblastine (50 microM) all abolished net secretion of [3H]-digoxin due to both a decrease in Jb-a and an increase in Ja-b. Cellular accumulation of [3H]-digoxin was also increased across both the apical and basal cell surfaces. I-Chloro-2,4,-dinitrobenzene (10 microM), a substrate for glutathione-S-transferase and subsequent ATP-dependent glutathione-S-conjugate secretion, failed to inhibit net secretion of [3H]-digoxin. The increase in absorptive permeability Pa-b (= Ja-b/Ca) and cellular [3H]-digoxin uptake upon P-glycoprotein inhibition, showed that the intestinal epithelium was rendered effectively impermeable by ATP-dependent extrusion at the apical surface. 5. A model for [3H]-digoxin secretion by the intestinal epithelium is likely to involve both diffusional uptake and Na(+)-K+ pump-mediated endocytosis, followed by active extrusion at the apical membrane. PMID:8832062

  18. IL-10-producing CD4+ T cells negatively regulate fucosylation of epithelial cells in the gut

    PubMed Central

    Goto, Yoshiyuki; Lamichhane, Aayam; Kamioka, Mariko; Sato, Shintaro; Honda, Kenya; Kunisawa, Jun; Kiyono, Hiroshi

    2015-01-01

    Fucosylated glycans on the surface of epithelial cells (ECs) regulate intestinal homeostasis by serving as attachment receptors and a nutrient source for some species of bacteria. We show here that epithelial fucosylation in the ileum is negatively regulated by IL-10-producing CD4+ T cells. The number of fucosylated ECs was increased in the ileum of mice lacking T cells, especially those expressing αβ T cell receptor (TCR), CD4, and IL-10. No such effect was observed in mice lacking B cells. Adoptive transfer of αβTCR+ CD4+ T cells from normal mice, but not IL-10-deficient mice, normalized fucosylation of ECs. These findings suggest that IL-10-producing CD4+ T cells contribute to the maintenance of the function of ECs by regulating their fucosylation. PMID:26522513

  19. IL-10-producing CD4(+) T cells negatively regulate fucosylation of epithelial cells in the gut.

    PubMed

    Goto, Yoshiyuki; Lamichhane, Aayam; Kamioka, Mariko; Sato, Shintaro; Honda, Kenya; Kunisawa, Jun; Kiyono, Hiroshi

    2015-01-01

    Fucosylated glycans on the surface of epithelial cells (ECs) regulate intestinal homeostasis by serving as attachment receptors and a nutrient source for some species of bacteria. We show here that epithelial fucosylation in the ileum is negatively regulated by IL-10-producing CD4(+) T cells. The number of fucosylated ECs was increased in the ileum of mice lacking T cells, especially those expressing ?? T cell receptor (TCR), CD4, and IL-10. No such effect was observed in mice lacking B cells. Adoptive transfer of ??TCR(+) CD4(+) T cells from normal mice, but not IL-10-deficient mice, normalized fucosylation of ECs. These findings suggest that IL-10-producing CD4(+) T cells contribute to the maintenance of the function of ECs by regulating their fucosylation. PMID:26522513

  20. Human Bronchial Epithelial Cell Response to Heavy Particle Exposure

    NASA Astrophysics Data System (ADS)

    Story, Michael; Ding, Liang-Hao; Minna, John; Park, Seong-mi; Peyton, Michael; Larsen, Jill

    2012-07-01

    A battery of non-oncogenically immortalized human bronchial epithelial cells (HBECs) are being used to examine the molecular changes that lead to lung carcinogenesis after exposure to heavy particles found in the free space environment. The goal is to ultimately identify biomarkers of radioresponse that can be used for prediction of carcinogenic risk for fatal lung cancer. Our initial studies have focused on the cell line HBEC3 KT and the isogenic variant HBEC3 KTR53, which overexpresses the RASv12 mutant and where p53 has been knocked down by shRNA, and is considered to be a more oncogenically progressed variant. We have previously described the response of HBEC3 KT at the cellular and molecular level, however, the focus here is on the rate of cellular transformation after HZE radiation exposure and the molecular changes in transformed cells. When comparing the two cell lines we find that there is a maximum rate of cellular transformation at 0.25 Gy when cells are exposed to 1 GeV Fe particles, and, for the HBEC3 KTR53 there are multiple pathways upregulated that promote anchorage independent growth including the mTOR pathway, the TGF-1 pathway, RhoA signaling and the ERK/MAPK pathway as early as 2 weeks after radiation. This does not occur in the HBEC3 KT cell line. Transformed HBEC3 KT cells do not show any morphologic or phenotypic changes when grown as cell cultures. HBEC3 KTR53 cells on the other hand show substantial changes in morphology from a cobblestone epithelial appearance to a mesenchymal appearance with a lack of contact inhibition. This epithelial to mesenchymal change in morphology is accompanied by the expression of vimentin and a reduction in the expression of E-cadherin, which are hallmarks of epithelial to mesenchymal transition. Interestingly, for HBEC3 KT transformed cells there are no mutations in the p53 gene, 2 of 15 clones were found to be heterozygous for the RASV12 mutation, and 3 of 15 clones expressed high levels of BigH3, a TGFB-responsive gene associated with loss of cell anchorage. There is also a range of aneuploidy amongst the transformed clones and ongoing chromosomal analysis by array-based comparative genomic hybridization has identified single or two copy loss of the tumor suppressor gene FHIT, in 8 of 15 transformed clones. This is accompanied by a 6-fold reduction, overall, in FHIT gene expression amongst the 15 clones under examination. Interestingly, in spite of these changes at the molecular level, when implanted subcutaneously into immune-compromised mice, the transformed clones from the HBEC3 KT cell line do not form tumors. This suggests that additional hits are required for oncogenesis, at least in a subcutaneous model, and/or, 2-D tissue culture models to not adequately reflect the underlying biology. We have therefore, begun to examine transformation in a 3-D tissue culture model, bronchocysts, where HBEC cells ultimately differentiate and stop cycling. We have shown that cells in 3-D have reduced gene expression of key DNA repair genes, and are less effective at repairing complex damage. We are now irradiating at dose rates as low as 0.2 cGy/min to test the notion of an inverse dose rate effect for carcinogenesis by HZE particles. In our early experiments we have shown that as the dose rate dropped from 20 cGy/min to 0.2 cGy/min, for the same total dose (0.25 and 0.50 Gy) an increasing percentage of bronchocysts become mis-shapen, suggesting that some cells within the cyst have de-differentiated and have reentered the cell cycle. We are now testing whether those cells are, in fact, cycling and wherther they are transformed by disaggregating the cyst and placing the cells into soft agar culture.

  1. Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers.

    PubMed

    Pegtel, Dirk M; Middeldorp, Jaap; Thorley-Lawson, David A

    2004-11-01

    Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo. Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo. In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo. Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation. Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia. A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus. We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection. We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant. Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo. This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection. PMID:15507648

  2. Streptococcus salivarius K12 Limits Group B Streptococcus Vaginal Colonization

    PubMed Central

    Patras, Kathryn A.; Wescombe, Philip A.; Rösler, Berenice; Hale, John D.; Tagg, John R.

    2015-01-01

    Streptococcus agalactiae (group B streptococcus [GBS]) colonizes the rectovaginal tract in 20% to 30% of women and during pregnancy can be transmitted to the newborn, causing severe invasive disease. Current routine screening and antibiotic prophylaxis have fallen short of complete prevention of GBS transmission, and GBS remains a leading cause of neonatal infection. We have investigated the ability of Streptococcus salivarius, a predominant member of the native human oral microbiota, to control GBS colonization. Comparison of the antibacterial activities of multiple S. salivarius strains by use of a deferred-antagonism test showed that S. salivarius strain K12 exhibited the broadest spectrum of activity against GBS. K12 effectively inhibited all GBS strains tested, including disease-implicated isolates from newborns and colonizing isolates from the vaginal tract of pregnant women. Inhibition was dependent on the presence of megaplasmid pSsal-K12, which encodes the bacteriocins salivaricin A and salivaricin B; however, in coculture experiments, GBS growth was impeded by K12 independently of the megaplasmid. We also demonstrated that K12 adheres to and invades human vaginal epithelial cells at levels comparable to GBS. Inhibitory activity of K12 was examined in vivo using a mouse model of GBS vaginal colonization. Mice colonized with GBS were treated vaginally with K12. K12 administration significantly reduced GBS vaginal colonization in comparison to nontreated controls, and this effect was partially dependent on the K12 megaplasmid. Our results suggest that K12 may have potential as a preventative therapy to control GBS vaginal colonization and thereby prevent its transmission to the neonate during pregnancy. PMID:26077762

  3. Streptococcus salivarius K12 Limits Group B Streptococcus Vaginal Colonization.

    PubMed

    Patras, Kathryn A; Wescombe, Philip A; Rösler, Berenice; Hale, John D; Tagg, John R; Doran, Kelly S

    2015-09-01

    Streptococcus agalactiae (group B streptococcus [GBS]) colonizes the rectovaginal tract in 20% to 30% of women and during pregnancy can be transmitted to the newborn, causing severe invasive disease. Current routine screening and antibiotic prophylaxis have fallen short of complete prevention of GBS transmission, and GBS remains a leading cause of neonatal infection. We have investigated the ability of Streptococcus salivarius, a predominant member of the native human oral microbiota, to control GBS colonization. Comparison of the antibacterial activities of multiple S. salivarius strains by use of a deferred-antagonism test showed that S. salivarius strain K12 exhibited the broadest spectrum of activity against GBS. K12 effectively inhibited all GBS strains tested, including disease-implicated isolates from newborns and colonizing isolates from the vaginal tract of pregnant women. Inhibition was dependent on the presence of megaplasmid pSsal-K12, which encodes the bacteriocins salivaricin A and salivaricin B; however, in coculture experiments, GBS growth was impeded by K12 independently of the megaplasmid. We also demonstrated that K12 adheres to and invades human vaginal epithelial cells at levels comparable to GBS. Inhibitory activity of K12 was examined in vivo using a mouse model of GBS vaginal colonization. Mice colonized with GBS were treated vaginally with K12. K12 administration significantly reduced GBS vaginal colonization in comparison to nontreated controls, and this effect was partially dependent on the K12 megaplasmid. Our results suggest that K12 may have potential as a preventative therapy to control GBS vaginal colonization and thereby prevent its transmission to the neonate during pregnancy. PMID:26077762

  4. Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence.

    PubMed

    Pratakpiriya, Watanyoo; Seki, Fumio; Otsuki, Noriyuki; Sakai, Kouji; Fukuhara, Hideo; Katamoto, Hiromu; Hirai, Takuya; Maenaka, Katsumi; Techangamsuwan, Somporn; Lan, Nguyen Thi; Takeda, Makoto; Yamaguchi, Ryoji

    2012-09-01

    Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence. PMID:22761370

  5. Small-Molecule Induction Promotes Corneal Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells

    PubMed Central

    Mikhailova, Alexandra; Ilmarinen, Tanja; Uusitalo, Hannu; Skottman, Heli

    2014-01-01

    Summary Human induced pluripotent stem cells (hiPSCs) offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF) in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation. PMID:24527395

  6. Epithelial cells are a source of natural IgM that contribute to innate immune responses.

    PubMed

    Shao, Wenwei; Hu, Fanlei; Ma, Junfan; Zhang, Chi; Liao, Qinyuan; Zhu, Zhu; Liu, Enyang; Qiu, Xiaoyan

    2016-04-01

    Currently, natural IgM antibodies are considered to be the constitutively secreted products of B-1 cells in mice and humans. In this study, we found that mouse epithelial cells, including liver epithelial cells and small intestinal epithelial cells (IECs), could express IgM that also showed natural antibody activity. Moreover, similar to the B-1 cell-derived natural IgM that can be upregulated by TLR9 agonists (mimicking bacterial infection), the expression of epithelial cell-derived natural IgM could also be significantly increased by TLR9 signaling. More importantly, the epithelial cell-derived IgM was polyreactive, and it could recognize single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), lipopolysaccharide (LPS), and insulin with low affinity; additionally, TLR9 agonists could enhance it in a MyD88-dependent manner. Furthermore, epithelial cell-derived IgM could bind various bacteria; therefore, it could be involved in anti-infection responses. Together, these results highlight the fact that epithelial cells are an important source of natural IgM, in addition to that produced by B-1 cells, and IgM contributes to the innate immune responses in local tissues, further demonstrating that the epithelium is a first line of defense in the protection against invading microbes. PMID:26820901

  7. Epithelial-mesenchymal transition and fibrosis are mutually exclusive reponses in shear-activated proximal tubular epithelial cells.

    PubMed

    Grabias, Bryan M; Konstantopoulos, Konstantinos

    2012-10-01

    Renal fibrosis (RF) is thought to be a direct consequence of dedifferentiation of resident epithelial cells via an epithelial-mesenchymal transition (EMT). Increased glomerular flow is a critical initiator of fibrogenesis. Yet, the responses of proximal tubular epithelial cells (PTECs) to fluid flow remain uncharacterized. Here, we investigate the effects of pathological shear stresses on the development of fibrosis in PTECs. Our data reveal that type I collagen accumulation in shear-activated PTECs is accompanied by a ∼40-60% decrease in cell motility, thus excluding EMT as a relevant pathological process. In contrast, static incubation of PTECs with TGFβ1 increases cell motility by ∼50%, and induces stable expression of key mesenchymal markers, including Snail1, N-cadherin, and vimentin. Ectopic expression of TGFβ1 in shear-activated PTECs fails to induce EMT-associated changes but abrogates collagen accumulation via SMAD2-dependent mechanisms. Shear-mediated inhibition of EMT occurs via cyclic oscillations in both ERK2 activity and downstream expression of EMT genes. A constitutive ERK2 mutant induces stable expression of Snail1, N-cadherin, and vimentin, and increases cell motility in shear-activated PTECs by 250% without concomitant collagen deposition. Collectively, our data reveal that RF not only occurs without EMT but also that these two responses represent mutually exclusive cell fates. PMID:22744866

  8. Particle characteristics responsible for effects on human lung epithelial cells.

    PubMed

    Aust, Ann E; Ball, James C; Hu, Autumn A; Lighty, JoAnn S; Smith, Kevin R; Straccia, Ann M; Veranth, John M; Young, Willie C

    2002-12-01

    Some recent epidemiologic investigations have shown an association between increased incidence of respiratory symptoms and exposure to low levels of particulate matter (PM*) less than 10 microm or less than 2.5 microm in aerodynamic diameter (PM10 and PM2.5, respectively). If particulates are causally involved with respiratory symptoms, it is important to understand which components may be responsible. However, increasing evidence suggests that transition metals present in particles, especially iron, generate reactive oxygen species (ROS) that may be involved in producing some of the observed respiratory symptoms. The hypothesis for this study is twofold: bioavailable transition metals from inhaled airborne particulates catalyze redox reactions in human lung epithelial cells, leading to oxidative stress and increased production of mediators of pulmonary inflammation: and the size, transition metal content, and mineral speciation of particulates affect their ability to cause these effects. This work focused on the relation between physical characteristics of particles (eg, size, bioavailable transition metal content, and mineral speciation) and their ability to generate hydroxyl radicals in cell-free systems and to cause oxidative stress, which results in the synthesis of mediators of pulmonary inflammation in cultured human lung epithelial cells. These relations were studied by comparing size-fractionated, chemically characterized coal fly ash (CFA) produced by combustion of three different coals to obtain milligram quantities of ash. One transition metal, iron, was studied specifically because it is by far the predominant transition metal in CFA. In addition, smaller quantities of particles from gasoline engines, diesel engines, and ambient air were studied. Phosphate buffer soluble fractions from particles from all sources were capable of generating ROS, as measured by production of malondialdehyde (MDA) from 2-deoxyribose. This activity was inhibited over 90% for all particles by the metal chelator N-[5-[3-[(5-aminopentyl)hydroxycarbamoyl]propionamidol-pentyl]-3-[[5-(N-hydroxyacetamido)pentyl]carbamoyl]propionohydroxamic acid (desferrioxamine B, or DF), strongly suggesting that transition metal(s), probably iron, were responsible. Particles from coal or gasoline combustion had greater ability to produce ROS than particles from diesel combustion. Iron was mobilized by citrate (at pH 7.5) from particles of all sources tested; gasoline combustion particles were the only particles not analyzed for iron mobilization because there were not enough particles for the iron mobilization assay. CFA particles were size-fractioned; the amount of iron mobilized by citrate was inversely related to the size of particles and also depended on the source of coal. Iron from the CFA particles was responsible for inducing the iron-storage protein ferritin in cultured human lung epithelial cells (A549 cells). The amount of iron mobilized by citrate was directly proportional to the amount of ferritin induced in the A549 cells. Iron from the CFA was also responsible for inducing the inflammatory mediator interleukin (IL) 8 in A549 cells. Iron existed in several species in the fly ash, but the bioavailable iron was associated with the glassy aluminosilicate fraction, which caused ferritin and IL-8 to be induced in the A549 cells. In crustal dust, another component of urban particulates, iron was associated with oxides and clay but not with aluminosilicates. The crustal dust contained almost no iron that could be mobilized by citrate. Iron could be mobilized from diesel combustion particulates, but at a much lower level than for all other combustion particles. Samples of ambient PM2.5 collected in Salt Lake City over 5-day periods during one month varied widely in the amount of iron that could be mobilized. If bioavailable transition metals (eg, iron) are related to the specific biological responses outlined here, then the potential exists to develop in vitro assays to determine whether particulates of unknown composition and origin can cause ef

  9. Upregulated CDK16 Expression in Serous Epithelial Ovarian Cancer Cells

    PubMed Central

    Zhou, Qi; Yu, Yanni

    2015-01-01

    Background As CDK-16 has been shown to be upregulated in several transformed cancer lines, we hypothesized that the cyclin-dependent kinase 16 (CDK-16) may be upregulated in serous epithelial ovarian cancer (EOC) cells. Therefore, we comparatively examined the mRNA and protein expression of CDK-16 in samples resected from serous EOC patients and normal controls. Material/Methods Tissue samples were collected from 70 serous EOC patients and 40 normal controls. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was conducted to assess mRNA expression. CDK-16 protein expression was assessed by semi-quantitative immunohistochemical staining. Differences in mRNA and protein expression between serous EOC cells and normal tissue cells were tested with the Kruskal-Wallis test and analysis of variance (ANOVA). Results Both CDK-16 mRNA and protein expression were significantly higher in serous EOC tumor cells as compared to normal control ovarian cells (p<0.01). Although there was no significant correlation between CDK-16 mRNA expression and serous EOC stage (p=0.0794), there was a significant correlation between CDK-16 mRNA expression and serous EOC grade (p<0.0001). Moreover, there were significant correlations between CDK-16 protein expression and serous EOC stage (p<0.0001) and grade (p<0.0001). Conclusions CDK-16 upregulation in serous EOC cells may represent a negative feedback loop to promote ovarian cell differentiation in malignantly-transformed serous EOC cells. Further in-depth investigation on CDK-16s role in serous EOC is needed. PMID:26546806

  10. Vaginal Fibroblastic Cells from Women with Pelvic Organ Prolapse Produce Matrices with Increased Stiffness and Collagen Content

    PubMed Central

    Ruiz-Zapata, Alejandra M.; Kerkhof, Manon H.; Ghazanfari, Samaneh; Zandieh-Doulabi, Behrouz; Stoop, Reinout; Smit, Theo H.; Helder, Marco N.

    2016-01-01

    Pelvic organ prolapse (POP) is characterised by the weakening of the pelvic floor support tissues, and often by subsequent prolapse of the bladder outside the body, i.e. cystocele. The bladder is kept in place by the anterior vaginal wall which consists of a dense extracellular matrix rich in collagen content that is maintained and remodelled by fibroblastic cells, i.e. fibroblasts and myofibroblasts. Since altered matrix production influences tissue quality, and myofibroblasts are involved in normal and pathological soft tissue repair processes, we evaluated matrix production of cells derived from pre- and post-menopausal POP and non-POP control anterior vaginal wall tissues. Results showed that cells from postmenopausal POP women deposited matrices with high percentage of collagen fibres with less anisotropic orientation and increased stiffness than those produced by controls. There was a transient increase in myofibroblastic phenotype that was lost after the peak of tissue remodelling. In conclusion, affected fibroblasts from postmenopausal prolapsed tissues produced altered matrices in vitro compared to controls. Such aberrant altered matrix production does not appear to be a consequence of abnormal phenotypical changes towards the myofibroblastic lineage. PMID:26965792

  11. Recruitment of Podocytes from Glomerular Parietal Epithelial Cells

    PubMed Central

    Appel, Daniel; Kershaw, David B.; Smeets, Bart; Yuan, Gang; Fuss, Astrid; Frye, Bjrn; Elger, Marlies; Kriz, Wilhelm; Floege, Jrgen; Moeller, Marcus J.

    2009-01-01

    Loss of a critical number of podocytes from the glomerular tuft leads to glomerulosclerosis. Even in health, some podocytes are lost into the urine. Because podocytes themselves cannot regenerate, we postulated that glomerular parietal epithelial cells (PECs), which proliferate throughout life and adjoin podocytes, may migrate to the glomerular tuft and differentiate into podocytes. Here, we describe transitional cells at the glomerular vascular stalk that exhibit features of both PECs and podocytes. Metabolic labeling in juvenile rats suggested that PECs migrate to become podocytes. To prove this, we generated triple-transgenic mice that allowed specific and irreversible labeling of PECs upon administration of doxycycline. PECs were followed in juvenile mice beginning from either postnatal day 5 or after nephrogenesis had ceased at postnatal day 10. In both cases, the number of genetically labeled cells increased over time. All genetically labeled cells coexpressed podocyte marker proteins. In conclusion, we demonstrate for the first time recruitment of podocytes from PECs in juvenile mice. Unraveling the mechanisms of PEC recruitment onto the glomerular tuft may lead to novel therapeutic approaches to renal injury. PMID:19092119

  12. Carbocisteine inhibits rhinovirus infection in human tracheal epithelial cells.

    PubMed

    Yasuda, H; Yamaya, M; Sasaki, T; Inoue, D; Nakayama, K; Yamada, M; Asada, M; Yoshida, M; Suzuki, T; Nishimura, H; Sasaki, H

    2006-07-01

    The aim of the study was to examine the effects of a mucolytic drug, carbocisteine, on rhinovirus (RV) infection in the airways. Human tracheal epithelial cells were infected with a major-group RV, RV14. RV14 infection increased virus titres and the cytokine content of supernatants. Carbocisteine reduced supernatant virus titres, the amount of RV14 RNA in cells, cell susceptibility to RV infection and supernatant cytokine concentrations, including interleukin (IL)-6 and IL-8, after RV14 infection. Carbocisteine reduced the expression of mRNA encoding intercellular adhesion molecule (ICAM)-1, the receptor for the major group of RVs. It also reduced the supernatant concentration of a soluble form of ICAM-1, the number and fluorescence intensity of acidic endosomes in the cells before RV infection, and nuclear factor-kappaB activation by RV14. Carbocisteine also reduced the supernatant virus titres of the minor group RV, RV2, although carbocisteine did not reduce the expression of mRNA encoding a low density lipoprotein receptor, the receptor for RV2. These results suggest that carbocisteine inhibits rhinovirus 2 infection by blocking rhinovirus RNA entry into the endosomes, and inhibits rhinovirus 14 infection by the same mechanism as well as by reducing intercellular adhesion molecule-1 levels. Carbocisteine may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection. PMID:16510461

  13. Isolation and characterization of a breast progenitor epithelial cell line with robust DNA damage responses.

    PubMed

    Shen, Kate C; Miller, Fred; Tait, Larry; Santner, Steven J; Pauley, Robert; Raz, Avraham; Tainsky, Michael A; Brooks, S C; Wang, Y Alan

    2006-08-01

    We report the establishment of a breast epithelial cell model that undergoes growth arrest at different stages of the cell cycle depending upon the DNA damaging agents encountered. Primary breast epithelial cells from normal reductive mammoplasty were grown in low-calcium culture medium. Free-floating cells under this condition were separated and used for establishment of the MCF-15 breast epithelial cell line. We found that MCF-15 breast epithelial cells display a superb response to different phases of the cell cycle arrest in response to various DNA damaging agents. Immunohistological analysis indicates that MCF-15 cells express cytokeratin 19, CD44, CXCR4, SDF-1, SPARC and vimentin. Although less than 5% of the MCF-15 cells expressed Muc-1 in culture, increased Muc-1 expression was observed in luminal epithelial cells along the newly formed lumen in xenografts. Furthermore, a small population of MCF-15 cells expressed estrogen receptor-alpha (ERalpha) in xenografts while ERalpha expression was not detected in monolayer culture. Therefore, the MCF-15 breast epithelial cell line possesses characteristics of breast progenitor cells and provides a good cell culture model for studying the response to DNA damage and the etiology of aggressive basal-like breast cancers. PMID:16541320

  14. Soluble Proteins Produced by Probiotic Bacteria Regulate Intestinal Epithelial Cell Survival and Growth

    PubMed Central

    YAN, FANG; CAO, HANWEI; COVER, TIMOTHY L.; WHITEHEAD, ROBERT; WASHINGTON, M. KAY; POLK, D. BRENT

    2011-01-01

    Background & Aims Increased inflammatory cytokine levels and intestinal epithelial cell apoptosis leading to disruption of epithelial integrity are major pathologic factors in inflammatory bowel diseases. The probiotic bacterium Lactobacillus rhamnosus GG (LGG) and factors recovered from LGG broth culture supernatant (LGG-s) prevent cytokine-induced apoptosis in human and mouse intestinal epithelial cells by regulating signaling pathways. Here, we purify and characterize 2 secreted LGG proteins that regulate intestinal epithelial cell antiapoptotic and proliferation responses. Methods LGG proteins were purified from LGG-s, analyzed, and used to generate polyclonal antibodies for immunodepletion of respective proteins from LGG-conditioned cell culture media (CM). Mouse colon epithelial cells and cultured colon explants were treated with purified proteins in the absence or presence of tumor necrosis factor (TNF). Akt activation, proliferation, tissue injury, apoptosis, and caspase-3 activation were determined. Results We purified 2 novel proteins, p75 (75 kilodaltons) and p40 (40 kilodaltons), from LGG-s. Each of these purified protein preparations activated Akt, inhibited cytokine-induced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. TNF-induced colon epithelial damage was significantly reduced by p75 and p40. Immunodepletion of p75 and p40 from LGG-CM reversed LGG-CM activation of Akt and its inhibitory effects on cytokine-induced apoptosis and loss of intestinal epithelial cells. Conclusions p75 and p40 are the first probiotic bacterial proteins demonstrated to promote intestinal epithelial homeostasis through specific signaling pathways. These findings suggest that probiotic bacterial components may be useful for preventing cytokine-mediated gastrointestinal diseases. PMID:17258729

  15. Acute respiratory bronchiolitis: an ultrastructural and autoradiographic study of epithelial cell injury and renewal in Rhesus monkeys exposed to ozone

    SciTech Connect

    Castleman, W.L.; Dungworth, D.L.; Schwartz, L.W.; Tyler, W.S.

    1980-03-01

    The pathogenesis of acute respiratory bronchiolitis was examined in Rhesus monkeys exposed to 0.8 ppM ozone for 4 to 50 hours. Epithelial injury and renewal were qualitatively and quantitatively characterized by correlated techniques of scanning and transmission electron microscopy as well as by light-microscopic autoradiography following labeling with tritiated thymidine. Extensive degeneration and necrosis of Type 1 epithelial cells occurred on the respiratory bronchiolar wall during the initial 4 to 12 hours of exposure. Increased numbers of labeled epithelial cells were present in this region after 18 hours of exposure, and the highest labeling index (18%) was measured after 50 hours of exposure. Most (67 to 80%) of the labeled cells and all the mitotic epithelial cells (22) observed ultrastructurally were cuboidal bronchiolar epithelial cells. Of the labeled epithelial cells, 20 to 33% were Type 2 epithelial cells. After 50 hours of exposure the respiratory bronchiolar epithelium was hyperplastic. The predominant inflammatory cell in respiratory bronchiolar exudate was the alveolar macrophage. Monkeys that were exposed for 50 hours and allowed to recover in unozonized air for 7 days had incomplete resolution of respiratory bronchiolar epithelial hyperplasia. The results indicate that Type 1 epithelial cells lining respiratory bronchioles are the cell types most sensitive to injury and that both cuboidal bronchiolar epithelial cells and Type 2 epithelial cells function as stem cells in epithelial renewal.

  16. Human intestinal epithelial cells express IL-10 through Toll-like receptor 4 (TLR4)-mediated epithelial-macrophage crosstalk

    PubMed Central

    Hyun, Jinhee; Romero, Laura; Riveron, Reldy; Flores, Claudia; Kanagavelu, Saravana; Chung, Kristina D.; Alonso, Ana; Sotolongo, John; Ruiz, Jose; Manukyan, Armine; Chun, Sally; Singh, Gaurav; Pedro, Salas; Targan, Stephan R.; Fukata, Masayuki

    2015-01-01

    In the intestine, interaction between epithelial cells and macrophages creates a unique immunoregulatory microenvironment necessary to maintain local immune and tissue homeostasis. Human intestinal epithelial cells (IECs) have been shown to express IL-10, which keeps epithelial integrity. We have demonstrated that bacterial signaling through TLR4 induces 15-Deoxy-Delta-12,14-prostaglandin J2 (15d-PGJ2) synthesis in intestinal macrophages by Cox-2 expression. Here we show that TLR4 signaling generates crosstalk between IECs and macrophages that enhances IL-10 expression in IECs. Direct stimulation of TLR4 leads to the expression of IL-10 in IECs, while the presence of macrophages in a transwell system induces another peak of IL-10 in IECs at a later time point. The second peak of the IL-10 expression is two times greater than the first peak. This late induction of IL-10 depends on a nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ that is accumulated in IECs by TLR4-mediated inhibition of the ubiquitin-proteasomal pathway. TLR4 signaling in macrophages in turn synthesizes 15d-PGJ2 through p38 and ERK activation and Cox-2 induction, which activates PPARγ in IECs. These results suggest that TLR4 signaling maintains IL-10 production in IECs by generating epithelial-macrophage crosstalk, which is an important mechanism in maintenance of intestinal homeostasis mediated through host-bacterial interactions. PMID:25171731

  17. The Par3/Par6/aPKC complex and epithelial cell polarity.

    PubMed

    Chen, Jia; Zhang, Mingjie

    2013-06-10

    Apical-basal polarity is the basic organizing principle of epithelial cells, and endows epithelial cells to function as defensive barriers and as mediators of vectorial transport of nutrients in and out of organisms. Apical-basal polarity is controlled by a number of conserved polarity factors that regulate cytoskeletal organizations, asymmetric distributions of cellular components, and directional transports across cells. Polarity factors often occupy specific membrane regions in response to the adhesion forces generated by cell-cell and cell-extracellular matrix interactions. Both internal polarity factors and the external extracellular matrices play fundamental roles in epithelial cell polarity establishment and maintenance. This review focuses on recent developments of the Par3/Par6/aPKC complex and its interacting proteins in epithelial cell polarity. PMID:23535009

  18. Curcumin Prevents Replication of Respiratory Syncytial Virus and the Epithelial Responses to It in Human Nasal Epithelial Cells

    PubMed Central

    Obata, Kazufumi; Kojima, Takashi; Masaki, Tomoyuki; Okabayashi, Tamaki; Yokota, Shinichi; Hirakawa, Satoshi; Nomura, Kazuaki; Takasawa, Akira; Murata, Masaki; Tanaka, Satoshi; Fuchimoto, Jun; Fujii, Nobuhiro; Tsutsumi, Hiroyuki; Himi, Tetsuo; Sawada, Norimasa

    2013-01-01

    The human nasal epithelium is the first line of defense during respiratory virus infection. Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma and severe lower respiratory tract disease in infants and young children. We previously reported in human nasal epithelial cells (HNECs), the replication and budding of RSV and the epithelial responses, including release of proinflammatory cytokines and enhancement of the tight junctions, are in part regulated via an NF-?B pathway. In this study, we investigated the effects of the NF-?B in HNECs infected with RSV. Curcumin prevented the replication and budding of RSV and the epithelial responses to it without cytotoxicity. Furthermore, the upregulation of the epithelial barrier function caused by infection with RSV was enhanced by curcumin. Curcumin also has wide pharmacokinetic effects as an inhibitor of NF-?B, eIF-2? dephosphorylation, proteasome and COX2. RSV-infected HNECs were treated with the eIF-2? dephosphorylation blocker salubrinal and the proteasome inhibitor MG132, and inhibitors of COX1 and COX2. Treatment with salubrinal, MG132 and COX2 inhibitor, like curcumin, prevented the replication of RSV and the epithelial responses, and treatment with salubrinal and MG132 enhanced the upregulation of tight junction molecules induced by infection with RSV. These results suggest that curcumin can prevent the replication of RSV and the epithelial responses to it without cytotoxicity and may act as therapy for severe lower respiratory tract disease in infants and young children caused by RSV infection. PMID:24058438

  19. Cytotoxic Effects of Curcumin in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Hollborn, Margrit; Chen, Rui; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas; Kohen, Leon

    2013-01-01

    Backround Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro. Methodology/Principal Findings Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 M and strongly reduced by curcumin above 50 M. Curcumin at 50 M increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 M) and delayed apoptosis (above 1 M). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. Conclusion It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (?10 M) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of cancer or in the treatment of eye diseases, retinal function should be monitored carefully. PMID:23555722

  20. Elastase induces lung epithelial cell autophagy through placental growth factor

    PubMed Central

    Hou, Hsin-Han; Cheng, Shih-Lung; Chung, Kuei-Pin; Kuo, Mark Yen-Ping; Yeh, Cheng-Chang; Chang, Bei-En; Lu, Hsuan-Hsuan; Wang, Hao-Chien; Yu, Chong-Jen

    2014-01-01

    Chronic obstructive pulmonary disease (COPD) is a devastating disease, which is associated with increasing mortality and morbidity. Therefore, there is a need to clearly define the COPD pathogenic mechanism and to explore effective therapies. Previous studies indicated that cigarette smoke (CS) induces autophagy and apoptosis in lung epithelial (LE) cells. Excessive ELANE/HNE (elastase, neutrophil elastase), a factor involved in protease-antiprotease imbalance and the pathogenesis of COPD, causes LE cell apoptosis and upregulates the expression of several stimulus-responsive genes. However, whether or not elastase induces autophagy in LE cell remains unknown. The level of PGF (placental growth factor) is higher in COPD patients than non-COPD controls. We hypothesize that elastase induces PGF expression and causes autophagy in LE cells. In this study, we demonstrated that porcine pancreatic elastase (PPE) induced PGF expression and secretion in LE cells in vitro and in vivo. The activation of MAPK8/JNK1 (mitogen-activated protein kinase 8) and MAPK14/p38alpha MAPK signaling pathways was involved in the PGF mediated regulation of the TSC (tuberous sclerosis complex) pathway and autophagy in LE cells. Notably, PGF-induced MAPK8 and MAPK14 signaling pathways mediated the inactivation of MTOR (mechanistic target of rapamycin), the upregulation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ?) and the increase of autophagosome formation in mice. Furthermore, the PPE-induced autophagy promotes further apoptosis in vitro and in vivo. In summary, elastase-induced autophagy promotes LE cell apoptosis and pulmonary emphysema through the upregulation of PGF. PGF and its downstream MAPK8 and MAPK14 signaling pathways are potential therapeutic targets for the treatment of emphysema and COPD. PMID:24988221

  1. Vaginal Memory T Cells Induced by Intranasal Vaccination Are Critical for Protective T Cell Recruitment and Prevention of Genital HSV-2 Disease

    PubMed Central

    Sato, Ayuko; Suwanto, Aldina; Okabe, Manami; Sato, Shintaro; Nochi, Tomonori; Imai, Takahiko; Koyanagi, Naoto; Kunisawa, Jun; Kawaguchi, Yasushi

    2014-01-01

    ABSTRACT Protective immunity against genital pathogens causing chronic infections, such as herpes simplex virus 2 (HSV-2) or human immunodeficiency virus, requires the induction of cell-mediated immune responses locally in the genital tract. Intranasal immunization with a thymidine kinase-deficient (TK?) mutant of HSV-2 effectively induces HSV-2-specific gamma interferon (IFN-?)-secreting memory T cell production and protective immunity against intravaginal challenge with wild-type HSV-2. However, the precise mechanism by which intranasal immunization induces protective immunity in the distant genital mucosa more effectively than does systemic immunization is unknown. Here, we showed that intranasal immunization with live HSV-2 TK? induced the production of effector T cells and their migration to, and retention in, the vaginal mucosa, whereas systemic vaccination barely established a local effector T cell pool, even when it induced the production of circulating memory T cells in the systemic compartment. The long-lasting HSV-2-specific local effector T cells induced by intranasal vaccination provided superior protection against intravaginal wild-type HSV-2 challenge by starting viral clearance at the entry site earlier than with intraperitoneal immunization. Intranasal immunization is an effective strategy for eliciting high levels of cell-mediated protection of the genital tract by providing long-lasting antigen (Ag)-specific local effector T cells without introducing topical infection or inflammation. IMPORTANCE Intranasal (i.n.) vaccines against sexually transmitted diseases that are caused by viruses such as herpes simplex virus 2 (HSV-2) have long been in development, but no vaccine candidate is currently available. Understanding the cellular mechanisms of immune responses in a distant vaginal mucosa induced by i.n. immunization with HSV-2 will contribute to designing such a vaccine. Our study demonstrated that i.n. immunization with an attenuated strain of HSV-2 generated long-lasting IFN-?-secreting T cells in vaginal mucosa more effectively than systemic immunization. We found that these vaginal effector memory T cells are critical for the early stage of viral clearance at natural infection sites and prevent severe vaginal inflammation and herpes encephalitis. PMID:25231301

  2. HUIEC, Human intestinal epithelial cell line with differentiated properties: process of isolation and characterisation.

    PubMed

    Gradisnik, Lidija; Trapecar, Martin; Rupnik, Marjan Slak; Velnar, Tomaz

    2015-12-01

    The intestinal epithelium is composed of diverse cell types, most abundant being the enterocytes. Among other functions, they maintain the intestinal barrier and play a critical role in the absorption of nutrients, drugs and toxins. This study describes the development and characterization of human intestinal epithelial cells (HUIEC), a spontaneously arising cell line established by selective trypsinization and cloning of the intestinal epithelium, resulti