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Sample records for a431 human epithelial

  1. Anticancer effects of cantharidin in A431 human skin cancer (Epidermoid carcinoma) cells in vitro and in vivo.

    PubMed

    Li, Chi-Chuan; Yu, Fu-Shun; Fan, Ming-Jen; Chen, Ya-Yin; Lien, Jin-Cherng; Chou, Yu-Cheng; Lu, Hsu-Feng; Tang, Nou-Ying; Peng, Shu-Fen; Huang, Wen-Wen; Chung, Jing-Gung

    2017-03-01

    Cantharidin (CTD), a potential anticancer agent of Traditional Chinese Medicine has cytotxic effects in different human cancer cell lines. The cytotoxic effects of CTD on A431 human skin cancer (epidermoid carcinoma) cells in vitro and in A431 cell xenograft mouse model were examined. In vitro, A431 human skin cell were treated with CTD for 24 and 48 h. Cell phase distribution, ROS production, Ca 2+ release, Caspase activity and the level of apoptosis associated proteins were measured. In vivo, A431 cell xenograft mouse model were examined. CTD-induced cell morphological changes and decreased percentage of viable A431 cells via G0/G1 phase arrest and induced apoptosis. CTD-induced G0/G1 phase arrest through the reduction of protein levels of cyclin E, CDK6, and cyclin D in A431 cells. CTD-induced cell apoptosis of A431 cells also was confirm by DNA gel electrophoresis showed CTD-induced DNA fragmentation. CTD reduced the mitochondrial membrane potential and stimulated release of cytochrome c, AIF and Endo G in A431 cells. Flow cytometry demonstrated that CTD increased activity of caspase-8, -9 and -3. However, when cells were pretreated with specific caspase inhibitors activity was reduced and cell viability increased. CTD increased protein levels of death receptors such as DR4, DR5, TRAIL and levels of the active form of caspase-8, -9 and -3 in A431 cells. AIF and Endo G proteins levels were also enhanced by CTD. In vivo studies showed that CTD significantly inhibited A431 cell xenograft tumors in mice. Taken together, these in vitro and in vivo results provide insight into the mechanisms of CTD on cell growth and tumor production. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 723-738, 2017. © 2016 Wiley Periodicals, Inc.

  2. The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata.

    PubMed

    Liu, B; Harrell, R; Lamb, D J; Dresden, M H; Spira, M

    1989-10-15

    Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25-2.0 Mrads is optimal for cell growth.

  3. Identification of specific gravity sensitive signal transduction pathways in human A431 carcinoma cells

    NASA Astrophysics Data System (ADS)

    Rijken, P. J.; de Groot, R. P.; Kruijer, W.; de Laat, S. W.; Verkleij, A. J.; Boonstra, J.

    Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive β-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate that EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

  4. Toxicity of dimethylmonothioarsinic acid toward human epidermoid carcinoma A431 cells.

    PubMed

    Naranmandura, Hua; Ibata, Kenji; Suzuki, Kazuo T

    2007-08-01

    Chronic ingestion of arsenic-contaminated drinking water induces skin lesions and urinary bladder cancer in humans. It is now recognized that thioarsenicals such as dimethylmonothioarsinic acid (DMMTA (V)) are commonly excreted in the urine of humans and animals and that the production of DMMTA (V) may be a risk factor for the development of the diseases caused by arsenic. The toxicity of DMMTA (V) was compared with that of related nonthiolated arsenicals with respect to cell viability, uptake ability, generation of reactive oxygen species (ROS), and cell cycle progression of human epidermoid carcinoma A431 cells, arsenate (iAs (V)), arsenite (iAs (III)), dimethylarsinic acid (DMA (V)), and dimethylarsinous acid (DMA (III)) being used as reference nonthiolated arsenicals. DMMTA (V) (LC 50 = 10.7 microM) was shown to be much more cytotoxic than iAs (V) (LC 50 = 571 microM) and DMA (V) (LC 50 = 843 microM), and its potency was shown to be close to that of trivalent arsenicals iAs (III) (LC 50 = 5.49 microM) and DMA (III) (LC 50 = 2.16 microM). The greater cytotoxicity of DMMTA (V) was associated with greater cellular uptake and distribution, and the level of intracellular ROS remarkably increased in A431 cells upon exposure to DMMTA (V) compared to that after exposure to other trivalent arsenicals at the respective LC 50. Exposure of DMMTA (V) to cells for 24 h induced cell cycle perturbation. Namely, the percentage of cells residing in S and G2/M phases increased from 10.2 and 15.6% to 46.5 and 20.8%, respectively. These results suggest that although DMMTA (V) is a pentavalent arsenical, it is taken up efficiently by cells and causes various levels of toxicity, in a manner different from that of nonthiolated pentavalent arsenicals, demonstrating that DMMTA (V) is one of the most toxic arsenic metabolites. The high cytotoxicity of DMMTA (V) was explained and/or proposed by (1) efficient uptake by cells followed by (2) its transformation to DMA (V), (3) producing ROS

  5. Purification of high-molecular-weight subfraction from porcine skin inhibiting proliferation of A431 human carcinoma epidermoid cells.

    PubMed

    Belova, O V; Sergienko, V I; Arion, V Ya; Lukanidina, T A; Moskvina, S N; Zimina, I V; Borisenko, G G; Lutsenko, G V; Grechikhina, M V; Kovaleva, E V; Klyuchnikova, Zh I

    2014-07-01

    Subfraction with a molecular weight >250 kDa isolated from porcine skin and inhibiting the proliferation of A431 human carcinoma epidermoid cells was purified by DEAE 32 anion exchange chromatography with NaCl concentration step-gradient. The effects of the initial subfraction and fractions obtained by separation in DEAE 32 on the proliferation of A431 human carcinoma epidermoid cells were studied in vitro in two tests (MTT and fluorescent test). The more sensitive fluorescent test showed the highest inhibitory activity of fraction No. 2 released from the column at 0.15 M NaCl. One major protein component and a series of minor protein components were detected in this fraction by vertical PAAG-SDS electrophoresis.

  6. Flurbiprofen benzyl nitrate (NBS-242) inhibits the growth of A-431 human epidermoid carcinoma cells and targets β-catenin.

    PubMed

    Nath, Niharika; Liu, Xiaoping; Jacobs, Lloydine; Kashfi, Khosrow

    2013-01-01

    The Wnt/β-catenin/T cell factor (TCF) signaling pathway is important in the development of nonmelanoma skin cancers (NMSCs). Nitric-oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are chemopreventive agents consisting of a traditional NSAID attached to an NO-releasing moiety through a chemical spacer. Previously we showed that an aromatic spacer enhanced the potency of a particular NO-NSAID compared to an aliphatic spacer. We synthesized an NO-releasing NSAID with an aromatic spacer (flurbiprofen benzyl nitrate, NBS-242), and using the human skin cancer cell line A-431, we evaluated its effects on cell kinetics, Wnt/β-catenin, cyclin D1, and caspase-3. NBS-242 inhibited the growth of A-431 cancer cells, being ~15-fold more potent than flurbiprofen and up to 5-fold more potent than NO-flurbiprofen with an aliphatic spacer, the half maximal inhibitory concentrations (IC50) for growth inhibition being 60 ± 4 μM, 320 ± 20 μM, and 880 ± 65 μM for NBS-242, NO-flurbiprofen, and flurbiprofen, respectively. This effect was associated with inhibition of proliferation, accumulation of cells in the G0/G1 phase of the cell cycle, and an increase in apoptotic cell population. NBS-242 cleaved β-catenin both in the cytoplasm and the nucleus of A-431 cells. NBS-242 activated caspase-3 whose activation was reflected in the cleavage of procaspase-3. To test the functional consequence of β-catenin cleavage, we determined the expression of cyclin D1, a Wnt-response gene. NBS-242 reduced cyclin D1 levels in a concentration dependent manner. These findings establish a strong inhibitory effect of NBS-242 in A-431 human epidermoid carcinoma cells. NBS-242 modulates parameters that are important in determining cellular mass.

  7. Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells

    SciTech Connect

    Madan, Esha; Prasad, Sahdeo; Roy, Preeti

    2008-12-26

    Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin present mainly in grapes, red wine and berries, is known to possess strong chemopreventive and anticancer properties. Here, we demonstrated the anti-proliferative and apoptosis-inducing activities of resveratrol in human epidermoid carcinoma A431 cells. Resveratrol has cytotoxic effects through inhibiting cellular proliferation of A431 cells, which leads to the induction of apoptosis, as evident by an increase in the fraction of cells in the sub-G{sub 1} phase of the cell cycle and Annexin-V binding of externalized phosphatidylserine. Results revealed that inhibition of proliferation is associated with regulation of the JAK/STAT pathway, where resveratrol prevents phosphorylation ofmore » JAK, thereby inhibiting STAT1 phosphorylation. Furthermore, resveratrol treatment actively stimulated reactive oxygen species (ROS) and mitochondrial membrane depolarization. Consequently, an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade and subsequent cleavage of PARP, thereby shifting the balance in favor of apoptosis. These observations indicate that resveratrol treatment inhibits JAK/STAT-mediated gene transcription and induce the mitochondrial cell death pathway.« less

  8. Fisetin inhibits growth, induces G2/M arrest and apoptosis of human epidermoid carcinoma A431 cells: Role of mitochondrial membrane potential disruption and consequent caspases activation

    PubMed Central

    Pal, Harish C.; Sharma, Samriti; Elmets, Craig A.; Athar, Mohammad; Afaq, Farrukh

    2013-01-01

    Non-melanoma skin cancers (NMSCs) one of the most common neoplasms causes serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3′,4′,7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and anti-proliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fistein (5-80 μM) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G2/M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad), (iii) disruption of mitochondrial potential, (iv) release of cytchrome c and Smac/DIABLO from mitochondria, (v) activation of caspases, and (vi) cleavage of PARP protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs. PMID:23800058

  9. A green approach toward quinoxalines and bis-quinoxalines and their biological evaluation against A431, human skin cancer cell lines.

    PubMed

    Bandyopadhyay, Debasish; Cruz, Jessica; Morales, Liza D; Arman, Hadi D; Cuate, Erica; Lee, Young S; Banik, Bimal K; Kim, Dae J

    2013-08-01

    The objective of this study was to develop a practical green procedure to synthesize quinoxalines and bis-quinoxalines and evaluate their inhibitory effects on the viability of A431 human epidermoid carcinoma cells. A series of quinoxaline and bis-quinoxaline derivatives have been designed and synthesized following a microwave-assisted and bismuth nitrate-catalyzed eco-friendly route. A detailed comparison has been made between microwave-induced protocol with the reactions occurred at room temperature. The structure of the compounds have been elucidated by various spectroscopic methods and finally confirmed by x-ray crystallographic analyses. Two quinoxaline derivatives, compounds 6 and 12 have demonstrated inhibitory effects on the viability of A431 human epidermoid carcinoma cells when compared with HaCaT nontumorigenic human keratinocyte cells. Notably, compound 6 inhibits Stat3 phosphorylation/activation in A431 skin cancer cells.

  10. Resveratrol enhances ultraviolet B-induced cell death through nuclear factor-{kappa}B pathway in human epidermoid carcinoma A431 cells

    SciTech Connect

    Roy, Preeti; Kalra, Neetu; Nigam, Nidhi

    Resveratrol has been reported to suppress cancer progression in several in vivo and in vitro models, whereas ultraviolet B (UVB), a major risk for skin cancer, is known to induce cell death in cancerous cells. Here, we investigated whether resveratrol can sensitize A431 human epidermoid carcinoma cells to UVB-induced cell death. We examined the combined effect of UVB (30 mJ/cm{sup 2}) and resveratrol (60 {mu}M) on A431 cells. Exposure of A431 carcinoma cells to UVB radiation or resveratrol can inhibit cell proliferation and induce apoptosis. However, the combination of resveratrol and UVB exposure was associated with increased proliferation inhibition ofmore » A431 cells compared with either agent alone. Furthermore, results showed that resveratrol and UVB treatment of A431 cells disrupted the nuclear factor-kappaB (NF-{kappa}B) pathway by blocking phosphorylation of serine 536 and inactivating NF-{kappa}B and subsequent degradation of I{kappa}B{alpha}, which regulates the expression of survivin. Resveratrol and UVB treatment also decreased the phosphorylation of tyrosine 701 of the important transcription factor signal transducer activator of transcription (STAT1), which in turn inhibited translocation of phospho-STAT1 to the nucleus. Moreover, resveratrol/UVB also inhibited the metastatic protein LIMK1, which reduced the motility of A431 cells. In conclusion, our study demonstrates that the combination of resveratrol and UVB act synergistically against skin cancer cells. Thus, resveratrol is a potential chemotherapeutic agent against skin carcinogenesis.« less

  11. A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR.

    PubMed

    Sun, Meng; Ren, Jing; Du, Hui; Zhang, Yanmin; Zhang, Jie; Wang, Sicen; He, Langchong

    2010-10-15

    We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

    PubMed Central

    2010-01-01

    Background α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action. Methods MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells. Results α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells. Conclusions This study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be

  13. Induction of apoptosis by lupeol in human epidermoid carcinoma A431 cells through regulation of mitochondrial, Akt/PKB and NFkappaB signaling pathways.

    PubMed

    Prasad, Sahdeo; Madan, Esha; Nigam, Nidhi; Roy, Preeti; George, Jasmine; Shukla, Yogeshwer

    2009-09-01

    The rising incidence of skin cancer in humans makes it equivalent to malignancies of organs. Therefore, it is necessary to intensify our efforts for better understanding and development of novel treatment and preventive approaches for skin cancer. Fruits and other plant derived products have gained considerable attention as they can reduce the risk of several cancer types. Lupeol, a triterpene, present in many fruits and medicinal plants, has been shown to possess many pharmacological properties including anti-cancer effect in both in vitro and in vivo assay systems. In the present study, apoptosis inducing effects of lupeol were studied in human epidermoid carcinoma A431 cells. Cell cycle analysis showed that lupeol treatment induces apoptosis (14-37%) in a dose-dependent manner as evident by an increased sub G(1) cell population. The RT-PCR and Western blot analysis showed that lupeol-induced apoptosis was associated with caspase dependent mitochondrial cell death pathway through activation of Bax, caspases, Apaf1, decrease in Bcl-2 expression and subsequent cleavage of PARP. Lupeol treatment also inhibited Akt/PKB signaling pathway by inhibition of Bad (Ser136) phosphorylation and 14-3-3 expression. In addition, lupeol treatment inhibited cell survival by inactivation of NFkappaB through upregulation of its inhibitor Ikappabetaalpha. The Caspase mediated apoptosis was noticed by decrease in lupeol induced apoptosis by Caspase inhibitors as well as increase in reactive oxygen species generation and loss of mitochondrial membrane potential. These results suggest that lupeol could be an effective anti-cancer agent and merits further investigation.

  14. [Primary culture of human normal epithelial cells].

    PubMed

    Tang, Yu; Xu, Wenji; Guo, Wanbei; Xie, Ming; Fang, Huilong; Chen, Chen; Zhou, Jun

    2017-11-28

    The traditional primary culture methods of human normal epithelial cells have disadvantages of low activity of cultured cells, the low cultivated rate and complicated operation. To solve these problems, researchers made many studies on culture process of human normal primary epithelial cell. In this paper, we mainly introduce some methods used in separation and purification of human normal epithelial cells, such as tissue separation method, enzyme digestion separation method, mechanical brushing method, red blood cell lysis method, percoll layered medium density gradient separation method. We also review some methods used in the culture and subculture, including serum-free medium combined with low mass fraction serum culture method, mouse tail collagen coating method, and glass culture bottle combined with plastic culture dish culture method. The biological characteristics of human normal epithelial cells, the methods of immunocytochemical staining, trypan blue exclusion are described. Moreover, the factors affecting the aseptic operation, the conditions of the extracellular environment, the conditions of the extracellular environment during culture, the number of differential adhesion, and the selection and dosage of additives are summarized.

  15. Bromelain inhibits nuclear factor kappa-B translocation, driving human epidermoid carcinoma A431 and melanoma A375 cells through G(2)/M arrest to apoptosis.

    PubMed

    Bhui, Kulpreet; Tyagi, Shilpa; Srivastava, Amit Kumar; Singh, Madhulika; Roy, Preeti; Singh, Richa; Shukla, Yogeshwer

    2012-03-01

    Bromelain, obtained from pineapple, is already in use clinically as adjunct in chemotherapy. Our objective was to test its ability to act as a sole anti-cancer agent. Therefore, we describe its anti-proliferative, anti-inflammatory and subsequent anti-cancer effects in vitro, against human epidermoid carcinoma-A431 and melanoma-A375 cells. Bromelain exhibited reduction in proliferation of both these cell-lines and suppressed their potential for anchorage-independent growth. Further, suppression of inflammatory signaling by bromelain was evident by inhibition of Akt regulated-nuclear factor-kappaB activation via suppression of inhibitory-kappaBα phosphorylation and concomitant reduction in cyclooxygenase-2. Since, the inflammatory cascade is well-known to be closely allied to cancer; we studied the effect of bromelain on events/molecules central to it. Bromelain caused depletion of intracellular glutathione and generation of reactive oxygen-species followed by mitochondrial membrane depolarization. This led to bromelain-induced cell-cycle arrest at G(2)/M phase which was mediated by modulation of cyclin B1, phospho-cdc25C, Plk1, phospho-cdc2, and myt1. This was subsequently followed by induction of apoptosis, indicated by membrane-blebbing, modulation of Bax-Bcl-2 ratio, Apaf-1, caspase-9, and caspase-3; chromatin-condensation, increase in caspase-activity and DNA-fragmentation. Bromelain afforded substantial anti-cancer potential in these settings; hence we suggest it as a potential prospect for anti-cancer agent besides only an additive in chemotherapy. Copyright ©2011 Wiley Periodicals, Inc.

  16. Interference of silibinin with IGF-1R signalling pathways protects human epidermoid carcinoma A431 cells from UVB-induced apoptosis

    SciTech Connect

    Liu, Weiwei; Otkur, Wuxiyar; Li, Lingzhi

    Highlights: ► Silibinin protects A431 cells from UVB irradiation-induced apoptosis. ► Up-regulation of the IGF-1R-JNK/ERK pathways by UVB induces cell apoptosis. ► Silibinin inhibits IGF-1R pathways to repress caspase-8-mediated apoptosis. -- Abstract: Ultraviolet B (UVB) from sunlight is a major cause of cutaneous lesion. Silibinin, a traditional hepatic protectant, elicits protective effects against UVB-induced cellular damage. In A431 cells, the insulin-like growth factor-1 receptor (IGF-1R) was markedly up-regulated by UVB irradiation. The activation of the IGF-1R signalling pathways contributed to apoptosis of the cells rather than rescuing the cells from death. Up-regulated IGF-1R stimulated downstream mitogen-activated protein kinases (MAPKs), suchmore » as c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases 1/2 (ERK1/2). The subsequent activation of caspase-8 and caspase-3 led to apoptosis. The activation of IGF-1R signalling pathways is the cause of A431 cell death. The pharmacological inhibitors and the small interfering RNA (siRNA) targeting IGF-1R suppressed the downstream activation of JNK/ERK-caspases to help the survival of the UVB-irradiated A431 cells. Indeed, silibinin treatment suppressed the IGF-1R-JNK/ERK pathways and thus protected the cells from UVB-induced apoptosis.« less

  17. 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

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

    PubMed Central

    Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, 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

  19. Leptin expression in human mammary epithelial cells and breast milk.

    PubMed

    Smith-Kirwin, S M; O'Connor, D M; De Johnston, J; Lancey, E D; Hassink, S G; Funanage, V L

    1998-05-01

    Leptin has recently been shown to be produced by the human placenta and potentially plays a role in fetal and neonatal growth. Many functions of the placenta are replaced by the mammary gland in terms of providing critical growth factors for the newborn. In this study, we show that leptin is produced by human mammary epithelial cells as revealed by RT/PCR analysis of total RNA from mammary gland and immunohistochemical staining of breast tissue, cultured mammary epithelial cells, and secretory epithelial cells present in human milk. We also verify that immunoreactive leptin is present in whole milk at 30- to 150-fold higher concentrations than skim milk. We propose that leptin is secreted by mammary epithelial cells in milk fat globules, which partition into the lipid portion of breast milk.

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

    PubMed

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

    2015-03-01

    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. Experimental laboratory study. University research institute. Epididymis from seven patients undergoing orchiectomy for suspected testicular cancer without epididymal involvement. Human epididymis epithelial cells harvested from adult epididymis tissue. Establishment of a robust culture protocol for adult human epididymal epithelial cells. 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. 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. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

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

    1994-10-01

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

  2. 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.

  3. Epithelial cells as alternative human biomatrices for comet assay.

    PubMed

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; 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.

  4. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; 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

  5. Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

    DTIC Science & Technology

    2005-07-01

    papilloma virus 16 E6 or E7. Proc. Nat. Acad. Sci. USA, 92: 3687-3691, 1995. 6. Huschtscha, L. I., Neumann, A. A., Noble, J. R., and Reddel, R. R. Effects...Oncology, In press. 5. Wazer, D. E., Liu, X.-L., Chu, Q., Gao, Q., and Band, V. Immortalization of distinct human mammary epithelial cell types by human

  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. Time-lapse recordings of human corneal epithelial healing.

    PubMed

    Hardarson, Thorir; Hanson, Charles; Claesson, Margareta; Stenevi, Ulf

    2004-04-01

    The aim of this study was to design an experimental set-up for the study of human corneal epithelial wound healing in a controlled in vitro situation. A time-lapse set-up was used. This allowed for pictures to be captured with a magnification ranging from x 80 to x 1800. Pictures were captured at 1-min intervals during the observation period, which lasted up to 4 days. Human corneal tissue was obtained from the Eye Bank or from surgery. A small, rounded lesion was produced in the corneal epithelium with a miniature drill. The specimens were placed in a mini-incubator; the camera focused on the epithelial lesion and continuously observed using the time-lapse set-up. The healing process of human corneal epithelium could be followed for several days. The initial healing response could be divided into a slow, a rapid and a consolidating phase. The first two phases lasted about 12 hours, and by then, epithelial cells covered the lesion. Depending on the origin of the tissue and the placement of the lesion, variations in the healing response could be seen. The time-lapse technique makes it possible to study epithelial wound healing over time at the cellular level. Data collected in this way can fill the gap between in vivo studies, where, by nature, human wound healing studies are restricted, and cell culture techniques, where cellular responses in many cases differ from the in vivo situation.

  8. Differential responses of Mcl-1 in photosensitized epithelial vs lymphoid-derived human cancer cells.

    PubMed

    Xue, Liang-yan; Chiu, Song-mao; Oleinick, Nancy L

    2005-10-20

    The antiapoptotic Bcl-2-family proteins, Bcl-2 and Bcl-xL, are recognized phototargets of photodynamic therapy (PDT) with the mitochondrion-targeting phthalocyanine photosensitizer Pc 4. In the present study, we found that myeloid cell leukemia 1 (Mcl-1), another antiapoptotic member of the Bcl-2 family, was not photodamaged in Pc 4-PDT-treated human carcinoma cells MCF-7c3, MDA-MB468, DU145, and A431, although Mcl-1 turnover was observed after exposure of HeLa or MCF-7c3 cells to a supralethal dose of UVC. In contrast, when human lymphoma U937 and Jurkat cells were treated with Pc 4-PDT, staurosporine (STS) or UVC, Mcl-1 was cleaved to generate a 28-kDa fragment over a 2-4 h period. The cleavage of Mcl-1 was accompanied by the activation of caspases-3, -9, and -8. The broad-specificity caspase inhibitor z-VAD-fmk completely blocked Mcl-1 cleavage induced by PDT, STS or UVC, providing evidence for Mcl-1 as a substrate for caspases. Western blot analysis localized Mcl-1 to mitochondria, ER, and cytosol of both MCF-7c3 and U937 cells, suggesting that Mcl-1 protein, unlike Bcl-2 and Bcl-xL, is not a target for Pc 4-PDT, probably due to its localization to sites removed from those of Pc 4 binding. The 28-kDa cleaved fragment of Mcl-1, which has proapoptotic activity, was produced in PDT-treated lymphoid-derived cells, but not in cells of epithelial origin, suggesting that PDT-induced rapid and extensive apoptosis in lymphoma cells may result in part from the sensitivity of their Mcl-1 to caspase cleavage, removing an important negative control on apoptosis.

  9. Establishment and Characterization of Immortalized Human Amniotic Epithelial Cells

    PubMed Central

    Zhou, Kaixuan; Koike, Chika; Yoshida, Toshiko; Okabe, Motonori; Fathy, Moustafa; Kyo, Satoru; Kiyono, Tohru; Saito, Shigeru

    2013-01-01

    Abstract Human amniotic epithelial cells (HAEs) have a low immunogenic profile and possess potent immunosuppressive properties. HAEs also have several characteristics similar to stem cells, and they are discarded after parturition. Thus, they could potentially be used in cell therapy with fewer ethical problems. HAEs have a short life, so our aim is to establish and characterize immortalized human amniotic epithelial cells (iHAEs). HAEs were introduced with viral oncogenes E6/E7 and with human telomerase reverse transcriptase (hTERT) to create iHAEs. These iHAEs have proliferated around 200 population doublings (PDs) for at least 12 months. High expression of stem cell markers (Oct 3/4, Nanog, Sox2, Klf4) and epithelial markers (CK5, CK18) were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). These iHAEs were expanded in ultra-low-attachment dishes to form spheroids similarly to epithelial stem/precursor cells. High expression of mesenchymal (CD44, CD73, CD90, CD105) and somatic (CD24, CD29, CD271, Nestin) stem cell markers was detected by flow cytometry. The iHAEs showed adipogenic, osteogenic, neuronal, and cardiac differentiation abilities. In conclusion, the immortalization of HAEs with the characteristics of stem cells has been established, allowing these iHAEs to become useful for cell therapy and regenerative medicine. PMID:23298399

  10. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.

    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 typemore » 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.« less

  11. 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

  12. Irsogladine maleate regulates gap junctional intercellular communication-dependent epithelial barrier in human nasal epithelial cells.

    PubMed

    Miyata, Ryo; Nomura, Kazuaki; Kakuki, Takuya; Takano, Ken-Ichi; Kohno, Takayuki; Konno, Takumi; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

    2015-04-01

    The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18β-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

  13. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2010-10-01

    This term reflects the method used to detect murine mammary stem cells which is based on their individual ability to regenerate an entire mammary tree......mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended

  14. [Characterization of epithelial primary culture from human conjunctiva].

    PubMed

    Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

    2014-01-01

    To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  15. Engineered human broncho-epithelial tissue-like assemblies

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor)

    2012-01-01

    Three-dimensional human broncho-epithelial tissue-like assemblies (TLAs) are produced in a rotating wall vessel (RWV) with microcarriers by coculturing mesenchymal bronchial-tracheal cells (BTC) and bronchial epithelium cells (BEC). These TLAs display structural characteristics and express markers of in vivo respiratory epithelia. TLAs are useful for screening compounds active in lung tissues such as antiviral compounds, cystic fibrosis treatments, allergens, and cytotoxic compounds.

  16. Stiffness nanotomography of human epithelial cancer cells

    NASA Astrophysics Data System (ADS)

    Staunton, Jack R.; Doss, Bryant L.; Gilbert, C. Michael; Kasas, Sandor; Ros, Robert

    2012-02-01

    The mechanical stiffness of individual cells is important in both cancer initiation and metastasis. We present atomic force microscopy (AFM) based nanoindentation experiments on various human mammary and esophagus cell lines covering the spectrum from normal immortalized cells to highly metastatic ones. The combination of an AFM with a confocal fluorescence lifetime imaging microscope (FLIM) in conjunction with the ability to move the sample and objective independently allow for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. This enables us to correlate the mechanical properties with the point of indentation in the FLIM image. We are using force-volume measurements as well as force indentation curves on distinct points on the cells to compare the elastic moduli of the nuclei, nucleoli, and the cytoplasm, and how they vary within and between individual cells and cell lines. Further, a detailed analysis of the force-indentation curves allows study of the cells' mechanical properties at different indentation depths and to generate 3D elasticity maps.

  17. Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells.

    PubMed

    Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Tan, Ay Eeng; Nur Azurah, Abdul Ghani; Hayati, Abdul Rahman

    2013-08-01

    The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50 ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10 ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-β1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

  18. Invasion of Human Oral Epithelial Cells by Prevotella intermedia

    PubMed Central

    Dorn, Brian R.; Leung, K.-P.; Progulske-Fox, Ann

    1998-01-01

    Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion. PMID:9826397

  19. The fate of epithelial cells in the human large intestine.

    PubMed

    Barkla, D H; Gibson, P R

    1999-08-01

    One hundred and forty biopsies of the colon and rectum, collected during routine colonoscopies of 51 patients aged 19 to 74 years, were examined using light microscopy and transmission and scanning electron microscopy. The results indicated that surface epithelial cells undergo apoptosis, passing through fenestrations in the basement membrane to where they enter the lamina propria and are taken up by macrophages; and it is hypothesized that apoptotic cells are carried through the fenestrations on a current of fluid. The study also found that epithelial cells positioned over the crypts are better attached and more robust than those more distant from the crypt opening; and it is further hypothesized that, after reaching the top of the crypts, some goblet cells cease secreting mucus and pass onto the surface compartment of absorptive cells. An unexpected finding was that the lower regions of the crypts commonly contain isolated necrotic colonocytes. Apoptotic cells were rarely observed in the crypt epithelium. The findings of this study support the "recycling" model of epithelial cell death in the surface compartment of the human colon.

  20. Characterization of an immortalized human vaginal epithelial cell line.

    PubMed

    Rajan, N; Pruden, D L; Kaznari, H; Cao, Q; Anderson, B E; Duncan, J L; Schaeffer, A J

    2000-02-01

    Adherence of type 1 piliated Escherichia coli to vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections (UTIs) in women. Progress in understanding the mechanism of adherence to the vaginal surface could be enhanced by the utilization of well-characterized vaginal epithelial cells. The objective of this study was to immortalize vaginal epithelial cells and study their bacterial adherence properties. Primary vaginal cells were obtained from a normal post-menopausal woman, immortalized by infection with E6/E7 genes from human papillomavirus 16 (HPV 16) and cultured in serum free keratinocyte growth factor medium. Positive immunostaining with a pool of antibodies to cytokeratins 1, 5, 10 and 14 (K1, K5, K10 and K14) and to K13 confirmed the epithelial origin of these cells. The immortalized cells showed binding of type 1 piliated E. coli in a pili specific and mannose sensitive manner. This model system should facilitate studies on the interaction of pathogens with vaginal mucosal cells, an essential step in the progression of ascending UTIs in women.

  1. 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.

  2. Bombesin-like peptide receptors in human bronchial epithelial cells.

    PubMed

    Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A

    1996-01-01

    Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

  3. 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

  4. [Wood smoke condensate induced epithelial-mesenchymal transition in human airway epithelial cells].

    PubMed

    Li, Wenxi; Zou, Weifeng; Li, Bing; Ran, Pixin

    2014-01-01

    To observe the detrimental effects of wood smoke condensate (WSC) exposure on human bronchial epithelial cells (HBEC), and to explore the expression of epithelial-mesenchymal transition (EMT) markers in HBEC exposed to WSC. HBEC were exposed respectively to 5, 10, 20, 40 and 50 mg/L of WSC /CSC for 7 days, with control groups only in cell culture medium at the same time, then the total cytoactivity was detected by cell counting kit-8. After observing the cellular morphology of WSC-stimulated HBEC. Western blot and immunofluorescence method were used to evaluate the expression levels of type I collagen, vimentin, E-cad and MMP-9 in HBEC exposed to WSC (10 mg/L) and cigarette smoke condensate (CSC) (10 mg/L) for 7 days. Statistical evaluation of the continuous data was performed by ANOVA. Independent-Samples t-test for between-group comparisons. After 7 days of exposure to WSC, HBEC manifested a morphological characteristic of loss of cell-cell contact and elongated shape. The level of E-cad was decreased in WSC exposure groups (Western blot: 0.30 ± 0.05, F = 22.07, P < 0.05) compared with the groups without WSC exposure (Western blot: 0.59 ± 0.08, F = 22.07, P < 0.05). In contrast, an upregulation in expression of type I collagen (Western blot: 0.58 ± 0.04 vs 0.26 ± 0.02, F = 119.72, P < 0.05) and MMP-9 (0.56 ± 0.08 vs 0.19 ± 0.03, F = 21.79, P < 0.05) was observed in the presence of WSC, compared with the control groups. Immunofluorescence analysis showed that after a 7-day exposure to WSC in these cells, the E-cad protein was lost whereas type I collagen, vimentin and MMP-9 were acquired. Both Western blot and immunofluorescence analysis showed no difference in expression levels of E-cad, type I collagen, vimentin and MMP-9 between WSC and CSC exposure groups. WSC exposure could induce EMT-like process in human airway epithelial cells.

  5. Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

    NASA Astrophysics Data System (ADS)

    Herceg, Zdenko

    Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the

  6. Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

    SciTech Connect

    Twite, Nicolas; Andrei, Graciela; Kummert, Caroline

    2014-07-15

    Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMVmore » by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.« less

  7. Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

    PubMed

    Yeung, Bonnie H Y; Wong, Chris K C

    2011-01-01

    Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

  8. Studying Mucin Secretion from Human Bronchial Epithelial Cell Primary Cultures

    PubMed Central

    Abdullah, Lubna H.; Wolber, Cédric; Kesimer, Mehmet; Sheehan, John K.; Davis, C. William

    2016-01-01

    Mucin secretion is regulated by extracellular signaling molecules emanating from local, neuronal, or endocrine sources. Quantifying the rate of this secretion is important to understanding how the exocytic process is regulated, and also how goblet/mucous cells synthesize and release mucins under control and pathological conditions. Consequently, measuring mucins in a quantitatively accurate manner is the key to many experiments addressing these issues. This paper describes procedures used to determine agonist-induced mucin secretion from goblet cells in human bronchial epithelial (HBE) cell cultures. It begins with primary epithelial cell culture, offers methods for purifying MUC5AC and MUC5B mucins for standards, and describes five different microtiter plate binding assays which use various probes for mucins. A polymeric mucin-specific antibody is used in standard and sandwich ELISA formats for two assays while the others target the extensive glycosylated domains of mucins with lectin, periodate oxidation, and antibody-based probes. Comparing the data derived from the different assays applied to the same set of samples of HBE cell cultures indicates a qualitative agreement between baseline and agonist stimulated mucin release; however, the polymeric mucin-specific assays yield substantially lower values than the assays using nonspecific molecular reporters. These results indicate that the more non-specific assays are suitable to assess overall secretory responses by goblet cells, but are likely unsuited for specific measurements of polymeric mucins, per se. PMID:22259142

  9. Proteomic and Bioinformatic Profile of Primary Human Oral Epithelial Cells

    PubMed Central

    Ghosh, Santosh K.; Yohannes, Elizabeth; Bebek, Gurkan; Weinberg, Aaron; Jiang, Bin; Willard, Belinda; Chance, Mark R.; Kinter, Michael T.; McCormick, Thomas S.

    2012-01-01

    Wounding of the oral mucosa occurs frequently in a highly septic environment. Remarkably, these wounds heal quickly and the oral cavity, for the most part, remains healthy. Deciphering the normal human oral epithelial cell (NHOEC) proteome is critical for understanding the mechanism(s) of protection elicited when the mucosal barrier is intact, as well as when it is breached. Combining 2D gel electrophoresis with shotgun proteomics resulted in identification of 1662 NHOEC proteins. Proteome annotations were performed based on protein classes, molecular functions, disease association and membership in canonical and metabolic signaling pathways. Comparing the NHOEC proteome with a database of innate immunity-relevant interactions (InnateDB) identified 64 common proteins associated with innate immunity. Comparison with published salivary proteomes revealed that 738/1662 NHOEC proteins were common, suggesting that significant numbers of salivary proteins are of epithelial origin. Gene ontology analysis showed similarities in the distributions of NHOEC and saliva proteomes with regard to biological processes, and molecular functions. We also assessed the inter-individual variability of the NHOEC proteome and observed it to be comparable with other primary cells. The baseline proteome described in this study should serve as a resource for proteome studies of the oral mucosa, especially in relation to disease processes. PMID:23035736

  10. 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

  11. 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...

  12. Culture models of human mammary epithelial cell transformation

    SciTech Connect

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

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

  13. 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.

  14. Cytotoxic effects of composite dust on human bronchial epithelial cells.

    PubMed

    Cokic, Stevan M; Hoet, Peter; Godderis, Lode; Wiemann, Martin; Asbach, Christof; Reichl, Franz X; De Munck, Jan; Van Meerbeek, Bart; Van Landuyt, Kirsten L

    2016-12-01

    Previous research revealed that during routine abrasive procedures like polishing, shaping or removing of composites, high amounts of respirable dust particles (<5μm) including nano-sized particles (<100nm) may be released. To determine the cytotoxic potential of composite dust particles on bronchial epithelium cells. Composite dust of five commercial composites (one nano-composite, two nano-hybrid and two hybrid composites) was generated following a clinically relevant protocol. Polymerized composite samples were cut with a rough diamond bur (grain size 100μm, speed 200,000rpm) and all composite dust was collected in a sterile chamber. Human bronchial epithelial cells (16HBE14o-) were exposed to serially diluted suspensions of composite dust in cell culture medium at concentrations between 1.1 and 3.3mg/ml. After 24h-exposure, cell viability and membrane integrity were assessed by the WST-1 and the LDH leakage assay, respectively. The release of IL-1β and IL-6 was evaluated. The composite dust particles were characterized by transmission electron microscopy and by dynamic and electrophoretic light scattering. Neither membrane damage nor release of IL-1β was detected over the complete concentration range. However, metabolic activity gradually declined for concentrations higher than 660μg/ml and the release of IL-6 was reduced when cells were exposed to the highest concentrations of dust. Composite dust prepared by conventional dental abrasion methods only affected human bronchial epithelial cells in very high concentrations. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  15. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

    PubMed Central

    Gudjonsson, Thorarinn; Villadsen, René; Nielsen, Helga Lind; Rønnov-Jessen, Lone; Bissell, Mina J.; Petersen, Ole William

    2002-01-01

    The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by

  16. Up-regulation of the tight-junction protein ZO-1 by substance P and IGF-1 in A431 cells.

    PubMed

    Ko, Ji-Ae; Murata, Shizuka; Nishida, Teruo

    2009-08-01

    The formation of a barrier by tight junctions is important in epithelia of various tissues. Substance P (SP) and insulin-like growth factor (IGF)-1 synergistically promote barrier function in the corneal epithelium. We have now examined the effects of SP and IGF-1 on expression of the tight-junction protein zonula occludens (ZO)-1 in A431 human epidermoid carcinoma cells. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analyses revealed that SP and IGF-1 increased the amounts of ZO-1 mRNA and protein in these cells in a concentration-dependent manner, with neither SP nor IGF-1 alone having such an effect. The SP- and IGF-1-induced up-regulation of ZO-1 was accompanied by phosphorylation of extracellular signal-regulated kinase (ERK), and both of these effects were blocked by PD98059, an inhibitor of ERK activation. SP and IGF-1 also increased the transepithelial electrical resistance (TER) (an indicator of barrier function) of an A431 cell monolayer in a manner sensitive to PD98059. Our results thus suggest that the synergistic induction of ZO-1 expression by SP and IGF-1 may promote barrier function in skin epithelial cells. (c) 2009 John Wiley & Sons, Ltd.

  17. Interleukin-1 stimulates zinc uptake by human thymic epithelial cells

    SciTech Connect

    Coto, J.A.; Hadden, J.W.

    1991-03-15

    Thymic epithelial cells (TEC) are known to secrete peptides which influence the differentiation and maturation of T-lymphocytes. These peptides include the thymic hormones thymulin, thymosin-{alpha}1, and thymopoietin. The biological activity of thymulin is dependent on the presence of zinc in an equimolar ratio. The authors have shown that both interleukin-1{alpha}(IL-1{alpha}) and interleukin-1{beta}(IL-1{beta}), which stimulate proliferation of TEC, stimulate the uptake of Zn-65 in-vitro independent of this proliferation. Mitomycin-C was used to inhibit the proliferation of TEC. Two other stimulators of proliferation of TEC, bovine pituitary extract (BPE) and epidermal growth factor (EGF), did not stimulate zinc uptake by the TECmore » independent of proliferation. They have also shown, utilizing in-situ hybridization, that IL-1 and zinc induce metallothionein(MT) mRNA expression in human thymic epithelial cells. The exact role of metallothionein is not clear, but it is thought to be involved in regulation of trace metal metabolism, especially in maintenance of zinc homeostasis. Their current hypothesis is that IL-1 stimulates uptake of zinc into the TEC, followed by its complexing with metallothionein. Zinc is then thought to be transferred from metallothionein to thymulin. Immunostaining, utilizing an antithymulin antibody and a fluoresceinated goat anti-rabbit second antibody, confirms the presence of thymulin in TEC and its dependence on zinc. Upon stimulation, thymulin is then secreted. Known stimulants for thymulin include progesterone, dexamethasone, estradiol, testosterone, and prolactin. None of these secretagogues increase zinc uptake, suggesting the priming of the zinc-thymulin complex is unrelated to the regulation of its secretion.« less

  18. Epithelial to mesenchymal transition in human endocrine islet cells

    PubMed Central

    Moreno-Amador, José Luis; Téllez, Noèlia; Marin, Sandra; Aloy-Reverté, Caterina; Semino, Carlos; Nacher, Montserrat

    2018-01-01

    Background β-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells. Objective To investigate whether EMT takes place in the endocrine non-β cells of human islets. Methodology Human islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer. Results Co-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-β-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers. Conclusions In adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a β-cell phenotype. PMID:29360826

  19. 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.

  20. 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.

  1. A taxonomy of epithelial human cancer and their metastases

    PubMed Central

    2009-01-01

    Background Microarray technology has allowed to molecularly characterize many different cancer sites. This technology has the potential to individualize therapy and to discover new drug targets. However, due to technological differences and issues in standardized sample collection no study has evaluated the molecular profile of epithelial human cancer in a large number of samples and tissues. Additionally, it has not yet been extensively investigated whether metastases resemble their tissue of origin or tissue of destination. Methods We studied the expression profiles of a series of 1566 primary and 178 metastases by unsupervised hierarchical clustering. The clustering profile was subsequently investigated and correlated with clinico-pathological data. Statistical enrichment of clinico-pathological annotations of groups of samples was investigated using Fisher exact test. Gene set enrichment analysis (GSEA) and DAVID functional enrichment analysis were used to investigate the molecular pathways. Kaplan-Meier survival analysis and log-rank tests were used to investigate prognostic significance of gene signatures. Results Large clusters corresponding to breast, gastrointestinal, ovarian and kidney primary tissues emerged from the data. Chromophobe renal cell carcinoma clustered together with follicular differentiated thyroid carcinoma, which supports recent morphological descriptions of thyroid follicular carcinoma-like tumors in the kidney and suggests that they represent a subtype of chromophobe carcinoma. We also found an expression signature identifying primary tumors of squamous cell histology in multiple tissues. Next, a subset of ovarian tumors enriched with endometrioid histology clustered together with endometrium tumors, confirming that they share their etiopathogenesis, which strongly differs from serous ovarian tumors. In addition, the clustering of colon and breast tumors correlated with clinico-pathological characteristics. Moreover, a signature was

  2. Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

    PubMed Central

    Hao, Weidong; Bernard, Katie; Patel, Nita; Ulbrandt, Nancy; Feng, Hui; Svabek, Catherine; Wilson, Susan; Stracener, Christina; Wang, Kathy; Suzich, JoAnn; Blair, Wade

    2012-01-01

    Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors. PMID:23035218

  3. Time-and Concentration-Dependent Cytotoxicity of Ricin in Human Lung Epithelial Cells

    DTIC Science & Technology

    2007-07-01

    lectin, ricin communis agglutinin, which is not directly cytotoxic but does have an affinity for red blood cells and can lead to agglutination and...Time- and Concentration-Dependent Cytotoxicity of Ricin in Human Lung Epithelial Cells Sharmaine Ramasamy and David Proll Human...Disease Control (CDC) Select Agent List. Using human small airway epithelial cells , this is the first study to investigate the time- and dose-dependent

  4. Cytokine Expression and Production by Purified Helicobacter pylori Urease in Human Gastric Epithelial Cells

    PubMed Central

    Tanahashi, Toshihito; Kita, Masakazu; Kodama, Tadashi; Yamaoka, Yoshio; Sawai, Naoki; Ohno, Tomoyuki; Mitsufuji, Shoji; Wei, Ya-Ping; Kashima, Kei; Imanishi, Jiro

    2000-01-01

    Cytokines have been proposed to play an important role in Helicobacter pylori-associated gastroduodenal diseases, but the exact mechanism of the cytokine induction remains unclear. H. pylori urease, a major component of the soluble proteins extracted from bacterial cells, is considered to be one of the virulence factors for the inflammation in the gastric mucosa that is produced in H. pylori infection. However, the response of human gastric epithelial cells to the stimulation of urease has not been investigated. In the present study, we used human gastric epithelial cells in a primary culture system and examined whether H. pylori urease stimulates the gastric epithelial cells to induce proinflammatory cytokines by reverse transcription-PCR and enzyme-linked immunosorbent assay. First, by using peripheral blood mononuclear cells (PBMC) and a gastric cancer cell line (MKN-45 cells), we confirmed the ability of purified H. pylori urease to induce the production of proinflammatory cytokines. Furthermore, we demonstrated that the human gastric epithelial cells produced interleukin-6 (IL-6) and tumor necrosis factor alpha, but not IL-8, following stimulation with purified urease. The patterns of cytokine induction differed among human PBMC, MKN-45 cells, and human gastric epithelial cells. These results suggest that the human gastric epithelial cells contribute to the induction of proinflammatory cytokines by the stimulation of H. pylori urease, indicating that the epithelial cells were involved in the mucosal inflammation that accompanied H. pylori infection. PMID:10639431

  5. The expression of Egfl7 in human normal tissues and epithelial tumors.

    PubMed

    Fan, Chun; Yang, Lian-Yue; Wu, Fan; Tao, Yi-Ming; Liu, Lin-Sen; Zhang, Jin-Fan; He, Ya-Ning; Tang, Li-Li; Chen, Guo-Dong; Guo, Lei

    2013-04-23

    To investigate the expression of Egfl7 in normal adult human tissues and human epithelial tumors.
 RT-PCR and Western blot were employed to detect Egfl7 expression in normal adult human tissues and 10 human epithelial tumors including hepatocellular carcinoma (HCC), lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, malignant glioma, ovarian cancer and renal cancer. Immunohistochemistry and cytoimmunofluorescence were subsequently used to determine the localization of Egfl7 in human epithelial tumor tissues and cell lines. ELISA was also carried out to examine the serum Egfl7 levels in cancer patients. In addition, correlations between Egfl7 expression and clinicopathological features as well as prognosis of HCC and breast cancer were also analyzed on the basis of immunohistochemistry results.
 Egfl7 was differentially expressed in 19 adult human normal tissues and was overexpressed in all 10 human epithelial tumor tissues. The serum Egfl7 level was also significantly elevated in cancer patients. The increased Egfl7 expression in HCC correlated with vein invasion, absence of capsule formation, multiple tumor nodes and poor prognosis. Similarly, upregulation of Egfl7 in breast cancer correlated strongly with TNM stage, lymphatic metastasis, estrogen receptor positivity, Her2 positivity and poor prognosis. 
 Egfl7 is significantly upregulated in human epithelial tumor tissues, suggesting Egfl7 to be a potential biomarker for human epithelial tumors, especially HCC and breast cancer.

  6. Nanoceria have no genotoxic effect on human lens epithelial cells

    NASA Astrophysics Data System (ADS)

    Pierscionek, Barbara K.; Li, Yuebin; Yasseen, Akeel A.; Colhoun, Liza M.; Schachar, Ronald A.; Chen, Wei

    2010-01-01

    There are no treatments for reversing or halting cataract, a disease of the structural proteins in the eye lens, that has associations with other age-related degenerative conditions such as Alzheimer's disease. The incidence of cataract and associated conditions is increasing as the average age of the population rises. Protein folding diseases are difficult to assess in vivo as proteins and their age-related changes are assessed after extraction. Nanotechnology can be used to investigate protein changes in the intact lens as well as for a potential means of drug delivery. Nanoparticles, such as cerium oxide (CeO2) which have antioxidant properties, may even be used as a means of treating cataract directly. Prior to use in treatments, nanoparticle genotoxicity must be tested to assess the extent of any DNA or chromosomal damage. Sister chromatid exchanges were measured and DNA damage investigated using the alkaline COMET assay on cultured human lens epithelial cells, exposed to 5 and 10 µg ml-1 of CeO2 nanoparticles (nanoceria). Nanoceria at these dosages did not cause any DNA damage or significant increases in the number of sister chromatid exchanges. The absence of genotoxic effects on lens cells suggests that nanoceria, in the doses and exposures tested in this study, are not deleterious to the eye lens and have the potential for use in studying structural alterations, in developing non-surgical cataract treatments and in investigating other protein folding diseases.

  7. Regulation of potassium transport in human lens epithelial cells.

    PubMed

    Lauf, Peter K; Warwar, Ronald; Brown, Thomas L; Adragna, Norma C

    2006-01-01

    The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1 mM) and bumetanide (5 microM) discriminated between the Na/K pump ( approximately 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) ( approximately 50%). Cl-replacement with nitrate or sulfamate revealed <10% residual [ouabain+bumetanide]-insensitive K-Cl cotransport (KCC). At 0.3-0.5 mM, NEM stimulated the Na/K pump by 2-fold independent of external Na, KCC between 2 and 4-fold, and abolished approximately 90% of NKCC. Calyculin-A, a serine/threonine protein phosphatase-1 inhibitor, did not affect NKCC but inhibited KCC, whereas 10 microM staurosporine, a serine/threonine kinase inhibitor, abolished NKCC, and stimulated KCC only when followed by NEM treatment. The tyrosine-kinase inhibitor genistein, at concentrations >100 microM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria.

  8. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

    2015-09-15

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. © 2015. Published by The Company of Biologists Ltd.

  9. Tonsil Epithelial Factors May Influence Oropharyngeal Human Immunodeficiency Virus Transmission

    PubMed Central

    Moutsopoulos, Niki M.; Nares, Salvador; Nikitakis, Nikolaos; Rangel, Zoila; Wen, Jie; Munson, Peter; Sauk, John; Wahl, Sharon M.

    2007-01-01

    Tonsil epithelium has been implicated in human immunodeficiency virus (HIV) pathogenesis, but its role in oral transmission remains controversial. To study characteristics of this tissue, which may influence susceptibility or resistance to HIV, we performed microarray analysis of the tonsil epithelium. Our data revealed that genes related to immune functions such as antibody production and antigen processing were increasingly expressed in tonsil compared with the epithelium of another oropharyngeal site, the gingival epithelium. Importantly, tonsil epithelium highly expressed genes associated with HIV entrapment and/or transmission, including the HIV co-receptor CXCR4 and the potential HIV-binding molecules FcRγIII, complement receptor 2, and various complement components. Immunohistochemical staining confirmed the increased presence of CXCR4 in the tonsil epithelium compared with multiple oral epithelial sites, particularly in basal and parabasal layers. This increased expression of molecules involved in viral recognition, binding, and entry may favor virus-epithelium interactions in an environment with reduced innate antiviral mechanisms. Specifically, secretory leukocyte protease inhibitor, an innate molecule with anti-HIV activity, was minimal in the tonsil epithelium, in contrast to oral mucosa. Collectively, our data suggest that increased expression of molecules associated with HIV binding and entry coupled with decreased innate antiviral factors may render the tonsil a potential site for oral transmission. PMID:17620369

  10. The relevance of human stem cell-derived organoid models for epithelial translational medicine

    PubMed Central

    Hynds, Robert E.; Giangreco, Adam

    2014-01-01

    Epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year. Despite this, most fundamental epithelial organ research fails to produce new therapies and mortality rates for epithelial organ diseases remain unacceptably high. In large part, this failure in translating basic epithelial research into clinical therapy is due to a lack of relevance in existing preclinical models. To correct this, new models are required that improve preclinical target identification, pharmacological lead validation, and compound optimization. In this review, we discuss the relevance of human stem cell-derived, three-dimensional organoid models for addressing each of these challenges. We highlight the advantages of stem cell-derived organoid models over existing culture systems, discuss recent advances in epithelial tissue-specific organoids, and present a paradigm for using organoid models in human translational medicine. PMID:23203919

  11. Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco-2) cells

    PubMed Central

    Cavet, M E; West, M; Simmons, N L

    1997-01-01

    Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 μM) or bromosulphophthalein (100 μM). Similarly tetraethylammonium and N-′methylnicotinamide (10 mM) were without effect. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4′-diisothiocyanostilbene-2-2′-disulphonic acid (DIDS, 400 μM). Net secretion of ciprofloxacin was partially inhibited by 100 μM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretion in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na+, Cl− or K+ in the bathing media. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia. PMID:9283689

  12. Proinflammatory effects of cookstove emissions on human bronchial epithelial cells.

    PubMed

    Hawley, B; Volckens, J

    2013-02-01

    Approximately half of the world's population uses biomass fuel for indoor cooking and heating. This form of combustion typically occurs in open fires or primitive stoves. Human exposure to emissions from indoor biomass combustion is a global health concern, causing an estimated 1.5 million premature deaths each year. Many 'improved' stoves have been developed to address this concern; however, studies that examine exposure-response with cleaner-burning, more efficient stoves are few. The objective of this research was to evaluate the effects of traditional and cleaner-burning stove emissions on an established model of the bronchial epithelium. We exposed well-differentiated, normal human bronchial epithelial cells to emissions from a single biomass combustion event using either a traditional three-stone fire or one of two energy-efficient stoves. Air-liquid interface cultures were exposed using a novel, aerosol-to-cell deposition system. Cellular expression of a panel of three pro-inflammatory markers was evaluated at 1 and 24 h following exposure. Cells exposed to emissions from the cleaner-burning stoves generated significantly fewer amounts of pro-inflammatory markers than cells exposed to emissions from a traditional three-stone fire. Particulate matter emissions from each cookstove were substantially different, with the three-stone fire producing the largest concentrations of particles (by both number and mass). This study supports emerging evidence that more efficient cookstoves have the potential to reduce respiratory inflammation in settings where solid fuel combustion is used to meet basic domestic needs. Emissions from more efficient, cleaner-burning cookstoves produced less inflammation in well-differentiated bronchial lung cells. The results support evidence that more efficient cookstoves can reduce the health burden associated with exposure to indoor pollution from the combustion of biomass. © 2012 John Wiley & Sons A/S.

  13. Regulation of human corneal epithelial mucins by rebamipide.

    PubMed

    Itoh, Shinsaku; Itoh, Kuni; Shinohara, Hisashi

    2014-02-01

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

  14. Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

    SciTech Connect

    Lasalvia, Maria; Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari; Castellani, Stefano

    The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalizedmore » airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity

  15. Detonation Nanodiamond Toxicity in Human Airway Epithelial Cells Is Modulated by Air Oxidation

    EPA Science Inventory

    Detonational nanodiamonds (DND), a nanomaterial with an increasing range of industrial and biomedical applications, have previously been shown to induce a pro-inflammatory response in cultured human airway epithelial cells (HAEC). We now show that surface modifications induced by...

  16. 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

  17. Constitutive expression of HCA(2) in human retina and primary human retinal pigment epithelial cells.

    PubMed

    Yu, Alice L; Birke, Kerstin; Lorenz, Reinhard L; Welge-Lussen, Ulrich

    2014-05-01

    HCA2, a receptor of β-hydroxybutyrate and niacin, has recently been described in mouse retina and immortalized human retinal pigment epithelial (RPE) cell lines. As HCA2 might be a pharmacologic target, e.g. in diabetic retinopathy, we studied its expression in human retina and primary human RPE cells. Paraffin sections of human retina and primary human RPE cells were obtained from human donor eyes. Expression of HCA2 in human retina was investigated by immunohistochemistry of paraffin sections and by RT-PCR. HCA2 expression in primary human RPE cells was examined by immunocytochemistry and by Western-blot analysis. Positive immunohistochemical staining for HCA2 was found in paraffin sections of human retina, and positive immunocytochemical staining for HCA2 in primary human RPE cells. RT-PCR analysis detected mRNA expression of HCA2 in human retina. The expression of HCA2 protein was found in primary human RPE cells. Based on these results, HCA2 appears to be constitutively expressed in human retina and in primary human RPE cells. Although its functional role is still unknown, HCA2 may be potentially involved in the pathogenesis of various retinopathies and may offer a new therapeutic target.

  18. Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity.

    PubMed

    Nguyen, Quy H; Pervolarakis, Nicholas; Blake, Kerrigan; Ma, Dennis; Davis, Ryan Tevia; James, Nathan; Phung, Anh T; Willey, Elizabeth; Kumar, Raj; Jabart, Eric; Driver, Ian; Rock, Jason; Goga, Andrei; Khan, Seema A; Lawson, Devon A; Werb, Zena; Kessenbrock, Kai

    2018-05-23

    Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer.

  19. Potential Role for a Carbohydrate Moiety in Anti-Candida Activity of Human Oral Epithelial Cells

    PubMed Central

    Steele, Chad; Leigh, Janet; Swoboda, Rolf; Ozenci, Hatice; Fidel, Paul L.

    2001-01-01

    Candida albicans is both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candida host defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of several Candida species in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candida activity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candida activity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candida activity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence of C. albicans to oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose- and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candida activity occurs exclusively with viable epithelial cells through contact with C. albicans by

  20. Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues.

    PubMed

    Lee, In-Seung; Cho, Dong-Hyuk; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji Hoon; Kim, Kwanil; Jung, Hee-Jae; Jang, Hyeung-Jin

    2018-02-01

    Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues. Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10 ng/ml) treatment for 4 h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay. Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB. The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.

  1. In vitro perforation of human epithelial carcinoma cell with antibody-conjugated biodegradable microspheres illuminated by a single 80 femtosecond near-infrared laser pulse

    PubMed Central

    Terakawa, Mitsuhiro; Tsunoi, Yasuyuki; Mitsuhashi, Tatsuki

    2012-01-01

    Pulsed laser interaction with small metallic and dielectric particles has been receiving attention as a method of drug delivery to many cells. However, most of the particles are attended by many risks, which are mainly dependent upon particle size. Unlike other widely used particles, biodegradable particles have advantages of being broken down and eliminated by innate metabolic processes. In this paper, the perforation of cell membrane by a focused spot with transparent biodegradable microspheres excited by a single 800 nm, 80 fs laser pulse is demonstrated. A polylactic acid (PLA) sphere, a biodegradable polymer, was used. Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres. The focused intensity was also simulated by the three-dimensional finite-difference time-domain method. Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients. The present method can open a new avenue, which is considered an efficient adherent for the selective perforation of cells which express the specific antigen on the cell membrane. PMID:22679375

  2. In vitro safety evaluation of human nasal epithelial cell monolayers exposed to carrageenan sinus wash.

    PubMed

    Ramezanpour, Mahnaz; Murphy, Jae; Smith, Jason L P; Vreugde, Sarah; Psaltis, Alkis James

    2017-12-01

    Carrageenans have shown to reduce the viral load in nasal secretions and lower the incidence of secondary infections in children with common cold. Despite the widespread use of carrageenans in topical applications, the effect of carrageenans on the sinonasal epithelial barrier has not been elucidated. We investigate the effect of different carrageenans on the sinonasal epithelial barrier and inflammatory response in vitro. Iota and Kappa carrageenan delivered in saline irrigation solutions applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells from chronic rhinosinusitis patients and controls. Epithelial barrier structure was assessed by measuring the transepithelial electrical resistance (TEER) and immunolocalization of F actin. Ciliary beat frequency (CBF), toxicity, and inflammatory response was studied. Kappa or Iota carrageenan in the different solutions was not toxic, did not have detrimental effects on epithelial barrier structure and CBF. Rather, application of Kappa carrageenan significantly increased TEER and suppressed interleukin 6 (IL-6) secretion in ALI cultures from CRS patients. Kappa or Iota carrageenan solution was safe and did not negatively affect epithelial barrier function. Kappa carrageenan increased TEER and decreased IL-6 production in CRS patients, indicating positive effects on epithelial barrier function in vitro. © 2017 ARS-AAOA, LLC.

  3. Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells

    PubMed Central

    Huff, Ryan D.; Hsu, Alan C-Y.; Nichol, Kristy S.; Jones, Bernadette; Knight, Darryl A.; Wark, Peter A. B.; Hansbro, Philip M.

    2017-01-01

    Introduction The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Materials and methods Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. Results HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Conclusions Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines. PMID:28863172

  4. TLR3-mediated NF-{kappa}B signaling in human esophageal epithelial cells.

    PubMed

    Lim, Diana M; Narasimhan, Sneha; Michaylira, Carmen Z; Wang, Mei-Lun

    2009-12-01

    Despite its position at the front line against ingested pathogens, very little is presently known about the role of the esophageal epithelium in host innate immune defense. As a key player in the innate immune response, Toll-like receptor (TLR) signaling has not been well characterized in human esophageal epithelial cells. In the present study, we investigated the inflammatory response and signaling pathways activated by TLR stimulation of human esophageal cells in vitro. Using quantitative RT-PCR, we profiled the expression pattern of human TLRs 1-10 in primary esophageal keratinocytes (EPC2), immortalized nontransformed esophageal keratinocytes (EPC2-hTERT), and normal human esophageal mucosal biopsies and found that TLRs 1, 2, 3, and 5 were expressed both in vivo and in vitro. Using the cytokine IL-8 as a physiological read out of the inflammatory response, we found that TLR3 is the most functional of the expressed TLRs in both primary and immortalized esophageal epithelial cell lines in response to its synthetic ligand polyinosinic polycytidylic acid [poly(I:C)]. Through reporter gene studies, we show that poly(I:C)-induced NF-kappaB activation is critical for the transactivation of the IL-8 promoter in vitro and that nuclear translocation of NF-kappaB occurs at an early time point following poly(I:C) stimulation of esophageal epithelial cells. Importantly, we also show that poly(I:C) stimulation induces the NF-kappaB-dependent esophageal epithelial expression of TLR2, leading to enhanced epithelial responsiveness of EPC2-hTERT cells to TLR2 ligand stimulation, suggesting an important regulatory role for TLR3-mediated NF-kappaB signaling in the innate immune response of esophageal epithelial cells. Our findings demonstrate for the first time that TLR3 is highly functional in the human esophageal epithelium and that TLR3-mediated NF-kappaB signaling may play an important regulatory role in esophageal epithelial homeostasis.

  5. Phenotypic characterization of collagen gel embedded primary human breast epithelial cells in athymic nude mice.

    PubMed

    Yang, J; Guzman, R C; Popnikolov, N; Bandyopadhyay, G K; Christov, K; Collins, G; Nandi, S

    1994-06-30

    We have developed a method to characterize the phenotypes and tumorigenicity of dissociated human breast epithelial cells. The dissociated cells were first embedded in collagen gels and subsequently transplanted subcutaneously in vivo in athymic nude mice. The transplantation of dissociated epithelial cells from reduction mammoplasties, presumed to be normal, always resulted in normal histomorphology. Epithelial cells were arranged as short tubular structures consisting of lumina surrounded by epithelial cells with an occasional more complex branching structure. These outgrowths were surrounded by intact basement membrane and were embedded in collagen gel that, at termination, contained collagenous stroma with fibroblasts and blood vessels. In contrast, transplantation of dissociated breast epithelial cells from breast cancer specimens resulted in outgrowths with an invasive pattern infiltrating the collagen gel as well as frank invasion into vascular space, nerves and muscles. These observations were made long before the subsequent palpable stage which resulted if left in the mouse for a long enough time. The dissociated human breast epithelial cells thus retained their intrinsic property to undergo morphogenesis to reflect their original phenotype when placed in a suitable environment, the collagen gel.

  6. Determining adaptive and adverse oxidative stress responses in human bronical epithelial cells exposed to zinc

    EPA Science Inventory

    Determining adaptive and adverse oxidative stress responses in human bronchial epithelial cells exposed to zincJenna M. Currier1,2, Wan-Yun Cheng1, Rory Conolly1, Brian N. Chorley1Zinc is a ubiquitous contaminant of ambient air that presents an oxidant challenge to the human lung...

  7. SEASONAL EFFECTS OF ULTRAFINE, FINE, AND COARSE PARTICULATE MATTER (PM) ON HUMAN PRIMARY AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    SEASONAL EFFECTS OF ULTRAFINE, FINE, AND COARSE PARTICULATE MATTER (PM) ON HUMAN PRIMARY AIRWAY EPITHELIAL CELLS

    Exposure of humans to PM results in increased mortality and morbidity. Recent toxicology studies have shown a number of pathophysiological pulmonary and car...

  8. GENE EXPRESSION PROFILING OF NORMAL HUMAN BRONCHIAL EPITHELIAL CELLS EXPOSED TO TRIVALENT ARSENICALS AND DIMETHYLTHIOARSINIC ACID

    EPA Science Inventory

    Lung is a major target for arsenic carcinogenesis in humans. However, the carcinogenic mode of action of arsenicals is unknown. We investigated, in human bronchial epithelial (BEAS2B) cells, the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsi...

  9. Oral focal epithelial hyperplasia: report of 3 cases with human papillomavirus DNA sequencing analysis.

    PubMed

    Gültekin, S E; Tokman Yildirim, Benay; Sarisoy, S

    2011-01-01

    Focal epithelial hyperplasia (FEH), or Heck's disease, is a benign proliferative viral infection of the oral mucosa that is related to Human Papil-lomavirus (HPV), mainly subtypes 13 and 32. Although this condition is known to exist in numerous populations and ethnic groups, the reported cases among Caucasians are relatively rare. It presents as asymptomatic papules or nodules on the oral mucosa, gingiva, tongue, and lips. Histopathologically, it is characterized by parakeratosis, epithelial hyperplasia, focal acanthosis, fusion, and horizontal outgrowth of epithelial ridges and the cells named mitozoids. The purpose of this case report was to present 3 cases of focal epithelial hyperplasia in a pediatric age group. Histopathological and clinical features of cases are discussed and DNA sequencing analysis is reported in which HPV 13, HPV 32, and HPV 11 genomes are detected.

  10. Initiation of oncogenic transformation in human mammary epithelial cells by charged particles

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

    Experimental studies have shown that high linear-energy transfer (LET) charged particles can be more effective than x-rays and gamma-rays in inducing oncogenic transformation in cultured cells and tumors in animals. Based on these results, experiments were designed and performed with an immortal human mammary epithelial cell line (H184B5), and several clones transformed by heavy ions were obtained. Cell fusion experiments were subsequently done, and results indicate that the transforming gene(s) is recessive. Chromosome analysis with fluorescence in situ hybridization (FISH) techniques also showed additional translocations in transformed human mammary epithelial cells. In addition, studies with these cell lines indicate that heavy ions can effectively induce deletion, break, and dicentrics. Deletion of tumor suppressor gene(s) and/or formation of translocation through DNA double strand breaks is a likely mechanism for the initiation of oncogenic transformation in human mammary epithelial cells.

  11. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

    PubMed Central

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  12. Pre-existing Epithelial Diversity in Normal Human Livers: A Tissue-tethered Cytometric Analysis in Portal/Periportal Epithelial Cells

    PubMed Central

    Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Maier, John; Specht, Susan; Castillo-Rama, Marcela; Lunz, John; Roysam, Badrinath; Michalopoulos, George; Demetris, Anthony J.

    2012-01-01

    Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BEC). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the Canals of Hering and/or metaplasia of pre-existing mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high resolution whole slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes pre-existed in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g. scatter plots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically-associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. Results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bi-potential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Conclusion Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable pre-existent hybrid epithelial diversity in normal human liver. This computationally-enabled tissue analysis approach offers much broader potential beyond the results presented here. PMID:23150208

  13. Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

    PubMed

    Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

    2013-12-01

    Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.

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

    PubMed Central

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

    2013-01-01

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

  15. Identification of a major sialoprotein in the glycocalyx of human visceral glomerular epithelial cells.

    PubMed Central

    Kerjaschki, D; Poczewski, H; Dekan, G; Horvat, R; Balzar, E; Kraft, N; Atkins, R C

    1986-01-01

    Glomerular visceral epithelial cells are endowed with a sialic acid-rich surface coat (the "glomerular epithelial polyanion"), which in rat tissue contains the sialoprotein podocalyxin. We have identified a major membrane sialoprotein in human glomeruli that is similar to rat podocalyxin in its sialic acid-dependent binding of wheat germ agglutinin and in its localization on the surface of glomerular epithelial and endothelial cells, as shown by immunoelectron microscopy, using the monoclonal antibody PHM5. Differences in the sialoproteins of the two species are indicated by the discrepancy of their apparent molecular weights in sodium dodecyl sulfate gels, by the lack of cross reactivity of their specific antibodies, and by the lack of homology of their proteolytic peptide maps. It is therefore possible that the human glomerular sialoprotein and rat podocalyxin are evolutionarily distinct, but have similar functions. Images PMID:3533998

  16. Effects of organophosphorus flame retardant TDCPP on normal human corneal epithelial cells: Implications for human health.

    PubMed

    Xiang, Ping; Liu, Rong-Yan; Li, Chao; Gao, Peng; Cui, Xin-Yi; Ma, Lena Q

    2017-11-01

    Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most detected organophosphorus flame retardants (OPFRs) in the environment, especially in indoor dust. Continuous daily exposure to TDCPP-containing dust may adversely impact human cornea. However, its detrimental effects on human corneal epithelium are largely unknown. In this study, we investigated the cell apoptosis in normal human corneal epithelial cells (HCECs) after TDCPP exposure and elucidated the underlying molecular mechanisms. Our data indicated a dose-dependent decrease of cell viability after TDCPP exposure with LC 50 at 202 μg/mL. A concentration-dependent apoptotic sign was observed in HCECs after exposing to ≥2 μg/mL TDCPP. Endoplasmic reticulum stress induction was evidenced by up-regulation of its biomarker genes (ATF-4, CHOP, BiP, and XBP1). Furthermore, alternation of Bcl-2/Bax expression, mitochondrial membrane potential loss, cellular ATP content decrease, and caspase-3 and -9 activity increase were observed after exposing to 2 or 20 μg/mL TDCPP. Taken together, the data implicated the involvement of endoplasmic reticulum stress in TDCPP-induced HCEC apoptosis, probably mediated by mitochondrial apoptotic pathway. Our findings showed TDCPP exposure induced toxicity to human cornea. Due to TDCPP's presence at high levels in indoor dust, further study is warranted to evaluate its health risk on human corneas. Published by Elsevier Ltd.

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

    SciTech Connect

    Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.

    1990-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1992-07-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  20. Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

    PubMed

    Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

    2016-01-01

    This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

  1. Human Rhinovirus Infection of Epithelial Cells Modulates Airway Smooth Muscle Migration.

    PubMed

    Shariff, Sami; Shelfoon, Christopher; Holden, Neil S; Traves, Suzanne L; Wiehler, Shahina; Kooi, Cora; Proud, David; Leigh, Richard

    2017-06-01

    Airway remodeling, a characteristic feature of asthma, begins in early life. Recurrent human rhinovirus (HRV) infections are a potential inciting stimulus for remodeling. One component of airway remodeling is an increase in airway smooth muscle cell (ASMC) mass with a greater proximity of the ASMCs to the airway epithelium. We asked whether human bronchial epithelial cells infected with HRV produced mediators that are chemotactic for ASMCs. ASMC migration was investigated using the modified Boyden Chamber and the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences Inc., San Diego, CA). Multiplex bead analysis was used to measure HRV-induced epithelial chemokine release. The chemotactic effects of CCL5, CXCL8, and CXCL10 were also examined. Supernatants from HRV-infected epithelial cells caused ASMC chemotaxis. Pretreatment of ASMCs with pertussis toxin abrogated chemotaxis, as did treatment with formoterol, forskolin, or 8-bromo-cAMP. CCL5, CXCL8, and CXCL10 were the most up-regulated chemokines produced by HRV-infected airway epithelial cells. When recombinant CCL5, CXCL8, and CXCL10 were used at levels found in epithelial supernatants, they induced ASMC chemotaxis similar to that seen with epithelial cell supernatants. When examined individually, CCL5 was the most effective chemokine in causing ASMC migration, and treatment of supernatant from HRV-infected epithelial cells with anti-CCL5 antibodies significantly attenuated ASMC migration. These findings suggest that HRV-induced CCL5 can induce ASMC chemotaxis and thus may contribute to the pathogenesis of airway remodeling in patients with asthma.

  2. Human Papillomaviruses; Epithelial Tropisms, and the Development of Neoplasia

    PubMed Central

    Egawa, Nagayasu; Egawa, Kiyofumi; Griffin, Heather; Doorbar, John

    2015-01-01

    Papillomaviruses have evolved over many millions of years to propagate themselves at specific epithelial niches in a range of different host species. This has led to the great diversity of papillomaviruses that now exist, and to the appearance of distinct strategies for epithelial persistence. Many papillomaviruses minimise the risk of immune clearance by causing chronic asymptomatic infections, accompanied by long-term virion-production with only limited viral gene expression. Such lesions are typical of those caused by Beta HPV types in the general population, with viral activity being suppressed by host immunity. A second strategy requires the evolution of sophisticated immune evasion mechanisms, and allows some HPV types to cause prominent and persistent papillomas, even in immune competent individuals. Some Alphapapillomavirus types have evolved this strategy, including those that cause genital warts in young adults or common warts in children. These strategies reflect broad differences in virus protein function as well as differences in patterns of viral gene expression, with genotype-specific associations underlying the recent introduction of DNA testing, and also the introduction of vaccines to protect against cervical cancer. Interestingly, it appears that cellular environment and the site of infection affect viral pathogenicity by modulating viral gene expression. With the high-risk HPV gene products, changes in E6 and E7 expression are thought to account for the development of neoplasias at the endocervix, the anal and cervical transformation zones, and the tonsilar crypts and other oropharyngeal sites. A detailed analysis of site-specific patterns of gene expression and gene function is now prompted. PMID:26193301

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

    PubMed

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

    2015-08-01

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

  4. Structure of neuro-endocrine and neuro-epithelial interactions in human foetal pancreas.

    PubMed

    Krivova, Yuliya; Proshchina, Alexandra; Barabanov, Valeriy; Leonova, Olga; Saveliev, Sergey

    2016-12-01

    In the pancreas of many mammals including humans, endocrine islet cells can be integrated with the nervous system components into neuro-insular complexes. The mechanism of the formation of such complexes is not clearly understood. The present study evaluated the interactions between the nervous system components, epithelial cells and endocrine cells in the human pancreas. Foetal pancreas, gestational age 19-23 weeks (13 cases) and 30-34 weeks (7 cases), were studied using double immunohistochemical labeling with neural markers (S100 protein and beta III tubulin), epithelial marker (cytokeratin 19 (CK19)) and antibodies to insulin and glucagon. We first analyse the structure of neuro-insular complexes using confocal microscopy and provide immunohistochemical evidences of the presence of endocrine cells within the ganglia or inside the nerve bundles. We showed that the nervous system components contact with the epithelial cells located in ducts or in clusters outside the ductal epithelium and form complexes with separate epithelial cells. We observed CK19-positive cells inside the ganglia and nerve bundles which were located separately or were integrated with the islets. Therefore, we conclude that neuro-insular complexes may forms as a result of integration between epithelial cells and nervous system components at the initial stages of islets formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation*

    PubMed Central

    Uchida, Hiroshi; Maruyama, Tetsuo; Nishikawa-Uchida, Sayaka; Oda, Hideyuki; Miyazaki, Kaoru; Yamasaki, Akiko; Yoshimura, Yasunori

    2012-01-01

    Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin. PMID:22174415

  6. ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO METALS

    EPA Science Inventory

    We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms respons...

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

    EPA Science Inventory

    Title: Asbestos-Induced Activation of Signaling Pathways in Human
    Bronchial Epithelial Cells

    X. Wang, MD 1, J. M. Samet, PhD 2 and A. J. Ghio, MD 2. 1 Center for
    Environmental Medicine, Asthma and Lung Biology, University of North
    Carolina, Chapel Hill, NC, Uni...

  8. THE EFFECT OF SIZE FRACTIONED PARTICULATE MATTER ON HUMAN AIRWAY EPITHELIAL CELLS IN VITRO

    EPA Science Inventory

    THE EFFECT OF SIZE FRACTIONATED PARTICULATE MATTER ON HUMAN AIRWAY EPITHELIAL CELLS IN VITRO. LA Dailey1, C Sioutas2, JM Soukup1, S Becker1, RB Devlin1. 1National Health & Environmental Effects Research Laboratory, USEPA, RTP, NC,USA; 2USC, Civil & Environmental Engineering, LA, ...

  9. SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES
    Y.M. Kim, A.G. Lenz, R. Silbajoris, I. Jaspers and J.M. Samet. Department of Environmental Sciences and Engineering and Center for Environmental Medicine, University of North Carolina, ...

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

    EPA Science Inventory

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

  11. DIFFERENTIAL ACTIVATION OF AP-1 IN HUMAN BLADDER EPITHELIAL CELLS BY INORGANIC AND METHYLATED ARSENICALS

    EPA Science Inventory

    Differential Activation of AP-1 in Human Bladder Epithelial Cells by Inorganic and Methylated Arsenicals

    Zuzana Drobna, Ilona Jaspers, David J. Thomas, and Miroslav Styblo

    ABSTRACT

    Epidemiological studies have linked chronic ingestion of drinking water contai...

  12. Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

    PubMed

    Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

    2007-11-23

    Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

  13. Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection.

    PubMed Central

    Lemoine, N. R.; Mayall, E. S.; Jones, T.; Sheer, D.; McDermid, S.; Kendall-Taylor, P.; Wynford-Thomas, D.

    1989-01-01

    Human primary thyroid follicular epithelial cells were transfected with a plasmid containing an origin-defective SV40 genome (SVori-) to produce several immortal cell lines. Two of the 10 cell lines analysed expressed specific features of thyroid epithelial function (iodide-trapping and thyroglobulin production). These two lines were characterised in detail and found to be growth factor-independent, capable of anchorage-independent growth at low frequency but non-tumorigenic in nude mice. These differentiated, These differentiated, partially transformed cell lines were shown to be suitable for gene transfer at high frequency using simple coprecipitation techniques. Images Figure 2 Figure 3 Figure 4 PMID:2557880

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

    PubMed Central

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

    2016-01-01

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

  15. In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

    PubMed

    Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2015-01-01

    Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue. © 2014 International Federation for Cell Biology.

  16. Disruption of β-catenin/CBP signaling inhibits human airway epithelial-mesenchymal transition and repair.

    PubMed

    Moheimani, Fatemeh; Roth, Hollis M; Cross, Jennifer; Reid, Andrew T; Shaheen, Furquan; Warner, Stephanie M; Hirota, Jeremy A; Kicic, Anthony; Hallstrand, Teal S; Kahn, Michael; Stick, Stephen M; Hansbro, Philip M; Hackett, Tillie-Louise; Knight, Darryl A

    2015-11-01

    The epithelium of asthmatics is characterized by reduced expression of E-cadherin and increased expression of the basal cell markers ck-5 and p63 that is indicative of a relatively undifferentiated repairing epithelium. This phenotype correlates with increased proliferation, compromised wound healing and an enhanced capacity to undergo epithelial-mesenchymal transition (EMT). The transcription factor β-catenin plays a vital role in epithelial cell differentiation and regeneration, depending on the co-factor recruited. Transcriptional programs driven by the β-catenin/CBP axis are critical for maintaining an undifferentiated and proliferative state, whereas the β-catenin/p300 axis is associated with cell differentiation. We hypothesized that disrupting the β-catenin/CBP signaling axis would promote epithelial differentiation and inhibit EMT. We treated monolayer cultures of human airway epithelial cells with TGFβ1 in the presence or absence of the selective small molecule ICG-001 to inhibit β-catenin/CBP signaling. We used western blots to assess expression of an EMT signature, CBP, p300, β-catenin, fibronectin and ITGβ1 and scratch wound assays to assess epithelial cell migration. Snai-1 and -2 expressions were determined using q-PCR. Exposure to TGFβ1 induced EMT, characterized by reduced E-cadherin expression with increased expression of α-smooth muscle actin and EDA-fibronectin. Either co-treatment or therapeutic administration of ICG-001 completely inhibited TGFβ1-induced EMT. ICG-001 also reduced the expression of ck-5 and -19 independent of TGFβ1. Exposure to ICG-001 significantly inhibited epithelial cell proliferation and migration, coincident with a down regulation of ITGβ1 and fibronectin expression. These data support our hypothesis that modulating the β-catenin/CBP signaling axis plays a key role in epithelial plasticity and function. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Characterization of immortalized human mammary epithelial cell line HMEC 2.6.

    PubMed

    Joshi, Pooja S; Modur, Vishnu; Cheng, JiMing; Robinson, Kathy; Rao, Krishna

    2017-10-01

    Primary human mammary epithelial cells have a limited life span which makes it difficult to study them in vitro for most purposes. To overcome this problem, we have developed a cell line that was immortalized using defined genetic elements, and we have characterized this immortalized non-tumorigenic human mammary epithelial cell line to establish it as a potential model system. human mammary epithelial cells were obtained from a healthy individual undergoing reduction mammoplasty at SIU School of Medicine. The cells were transduced with CDK4R24C followed by transduction with human telomerase reverse transcriptase. Post all manipulation, the cells displayed a normal cell cycle phase distribution and were near diploid in nature, which was confirmed by flow cytometry and karyotyping. In vitro studies showed that the cells were anchorage dependent and were non-invasive in nature. The cell line expressed basal epithelial markers such as cytokeratin 7, CD10, and p63 and was negative for the expression of estrogen receptor and progesterone receptor. Upon G-band karyotyping, the cell line displayed the presence of a few cytogenic abnormalities, including trisomy 20 and trisomy 7, which are also commonly present in other immortalized mammary cell lines. Furthermore, the benign nature of these cells was confirmed by multiple in vitro and in vivo experiments. Therefore, we think that this cell line could serve as a good model to understand the molecular mechanisms involved in the development and progression of breast cancer and to also assess the effect of novel therapeutics on human mammary epithelial cells.

  18. Retinal Pigment Epithelial Cells are a Potential Reservoir for Ebola Virus in the Human Eye

    PubMed Central

    Smith, Justine R.; Todd, Shawn; Ashander, Liam M.; Charitou, Theodosia; Ma, Yuefang; Yeh, Steven; Crozier, Ian; Michael, Michael Z.; Appukuttan, Binoy; Williams, Keryn A.; Lynn, David J.; Marsh, Glenn A.

    2017-01-01

    Purpose Success of Ebola virus (EBOV) as a human pathogen relates at the molecular level primarily to blockade the host cell type I interferon (IFN) antiviral response. Most individuals who survive Ebola virus disease (EVD) develop a chronic disease syndrome: approximately one-quarter of survivors suffer from uveitis, which has been associated with presence of EBOV within the eye. Clinical observations of post-Ebola uveitis indicate involvement of retinal pigment epithelial cells. Methods We inoculated ARPE-19 human retinal pigment epithelial cells with EBOV, and followed course of infection by immunocytochemistry and measurement of titer in culture supernatant. To interrogate transcriptional responses of infected cells, we combined RNA sequencing with in silico pathway, gene ontology, transcription factor binding site, and network analyses. We measured infection-induced changes of selected transcripts by reverse transcription-quantitative polymerase chain reaction. Results Human retinal pigment epithelial cells were permissive to infection with EBOV, and supported viral replication and release of virus in high titer. Unexpectedly, 28% of 560 upregulated transcripts in EBOV-infected cells were type I IFN responsive, indicating a robust type I IFN response. Following EBOV infection, cells continued to express multiple immunomodulatory molecules linked to ocular immune privilege. Conclusions Human retinal pigment epithelial cells may serve as an intraocular reservoir for EBOV, and the molecular response of infected cells may contribute to the persistence of live EBOV within the human eye. Translational Relevance This bedside-to-bench research links ophthalmic findings in survivors of EVD who suffer from uveitis with interactions between retinal pigment epithelial cells and EBOV. PMID:28721309

  19. Lipid deposits and lipo-mucosomes in human cholecystitis and epithelial metaplasia in chronic cholecystitis.

    PubMed

    Gilloteaux, Jacques; Tomasello, Lisa M; Elgison, Deborah A

    2003-01-01

    Among the inflammatory changes seen in cholecystitis, the ultrastructural alterations of the human gallbladder epithelium include lipid and lipofuscin deposits, fusions of lipid deposits and mucus-containing vesicles forming complex substructural formations called lipo-mucosomes, and microvillar changes of sparse microvilli and basal bodies. Small, lipid-laden structures, such as VLDL-like vesicles, also are fused with the mucus vesicles. Epithelial cell sloughing could liberate and add lipo-mucosomes to the biliary sludge and participate in gallstone formation. With chronic cholelithiasis, fatty degeneration of scattered epithelial cells appears to alter the epithelial lining and favors metaplastic change that could lead to other pathologic changes, including carcinoma in situ-like lesions. In addition to lipid deposition in macrophages, lipid is also incorporated in other cells and tissues of the gallbladder wall (endothelium of capillaries, smooth muscles and fibrocytes).

  20. Oxytetracycline Inhibits Mucus Secretion and Inflammation in Human Airway Epithelial Cells.

    PubMed

    Shah, Said Ahmad; Ishinaga, Hajime; Takeuchi, Kazuhiko

    2017-01-01

    Oxytetracycline is a broad-spectrum antibiotic, but its nonantibacterial effects in the human respiratory tract are unknown. In this study, the effects of oxytetracycline on mucus secretion and inflammation were examined by PCR and ELISA in the human airway epithelial cell line NCI-H292. Oxytetracycline (10 μg/mL) significantly inhibited TNF-α-induced MUC5AC gene expression and MUC5AC protein levels in NCI-H292 cells. It also downregulated IL-8 and IL-1β gene expression and IL-1β protein levels. Our findings demonstrated that oxytetracycline suppressed mucus production and inflammation in human respiratory epithelial cells, providing further evidence for the usefulness of oxytetracycline for human airway inflammatory diseases. © 2017 S. Karger AG, Basel.

  1. Platelets are a possible regulator of human endometrial re-epithelialization during menstruation.

    PubMed

    Suginami, Koh; Sato, Yukiyasu; Horie, Akihito; Matsumoto, Hisanori; Kyo, Satoru; Araki, Yoshihiko; Konishi, Ikuo; Fujiwara, Hiroshi

    2017-01-01

    The human endometrium periodically breaks down and regenerates. As platelets have been reported to contribute to the tissue remodeling process, we examined the possible involvement of platelets in endometrial regeneration. The distribution of extravasating platelets throughout the menstrual cycle was immunohistochemically examined using human endometrial tissues. EM-E6/E7/hTERT cells, a human endometrial epithelial cell-derived immortalized cell line, were co-cultured with platelets, and the effects of platelets on the epithelialization response of EM-E6/E7/hTERT cells were investigated by attachment and permeability assays, immunohistochemical staining, and Western blot analysis. Immunohistochemical study showed numerous extravasated platelets in the subluminar stroma during the menstrual phase. The platelets promoted the cell-to-matrigel attachment of EM-E6/E7/hTERT cells concomitantly with the phosphorylation of focal adhesion kinase. They also promoted cell-to-cell contact among EM-E6/E7/hTERT cells in parallel with E-cadherin expression. These results indicate the possible involvement of platelets in the endometrial epithelial re-epithelialization process. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. XB130 translocation to microfilamentous structures mediates NNK-induced migration of human bronchial epithelial cells.

    PubMed

    Wu, Qifei; Nadesalingam, Jeya; Moodley, Serisha; Bai, Xiaohui; Liu, Mingyao

    2015-07-20

    Cigarette smoking contributes to the pathogenesis of chronic obstructive pulmonary disease and lung cancer. Nicotine-derived nitrosamine ketone (NNK) is the most potent carcinogen among cigarette smoking components, and is known to enhance migration of cancer cells. However, the effect of NNK on normal human bronchial epithelial cells is not well studied. XB130 is a member of actin filament associated protein family and is involved in cell morphology changes, cytoskeletal rearrangement and outgrowth formation, as well as cell migration. We hypothesized that XB130 mediates NNK-induced migration of normal human bronchial epithelial cells. Our results showed that, after NNK stimulation, XB130 was translocated to the cell periphery and enriched in cell motility-associated structures, such as lamellipodia, in normal human bronchial epithelial BEAS2B cells. Moreover, overexpression of XB130 significantly enhanced NNK-induced migration, which requires both the N- and C-termini of XB130. Overexpression of XB130 enhanced NNK-induced protein tyrosine phosphorylation and promoted matrix metalloproteinase-14 translocation to cell motility-associated cellular structures after NNK stimulation. XB130-mediated NNK-induced cell migration may contribute to airway epithelial repair; however, it may also be involved in cigarette smoking-related chronic obstructive pulmonary disease and lung cancer.

  3. Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

    PubMed Central

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

    2013-01-01

    Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

  4. In vitro epithelial differentiation of human induced pluripotent stem cells for vocal fold tissue engineering.

    PubMed

    Imaizumi, Mitsuyoshi; Sato, Yuka; Yang, David T; Thibeault, Susan L

    2013-12-01

    We determined the feasibility and optimization of differentiating human induced pluripotent stem cells (hiPS) into nonkeratinized stratified squamous epithelial cells for vocal fold engineering. hiPS were cultured and assessed for differentiation in 3 conditions: a 3-dimensional (3D) hyaluronic acid (HA) hydrogel scaffold, a 3D HA hydrogel scaffold with epidermal growth factor (EGF), and a 3D HA hydrogel scaffold cocultured with human vocal fold fibroblasts (hVFF). After 1, 2, and 4 weeks of cultivation, hiPS were selected for histology, immunohistochemistry, and/or transcript expression analysis. At 4 weeks, hiPS cultivated with hVFF or with EGF had significantly decreased levels of Oct 3/4, indicating loss of pluripotency. Immunofluorescence revealed the presence of pancytokeratin and of cytokeratin (CK) 13 and 14 epithelial-associated proteins at 4 weeks after cultivation in hiPS EGF and hiPS hVFF cultures. The transcript expression level of CK14 was significantly increased for hiPS hVFF cultures only and was measured concomitantly with cell morphology that was clearly cohesive and displayed a degree of nuclear polarity suggestive of epithelial differentiation. We found that hiPS cultivated in 3D HA hydrogel with hVFF demonstrated the most robust conversion evidence to date of epithelial differentiation. Further work is necessary to focus on amplification of these progenitors for application in vocal fold regenerative biology.

  5. TLR-Dependent Human Mucosal Epithelial Cell Responses to Microbial Pathogens

    PubMed Central

    McClure, Ryan; Massari, Paola

    2014-01-01

    Toll-like receptor (TLR) signaling represents one of the best studied pathways to implement defense mechanisms against invading microbes in human being as well as in animals. TLRs respond to specific microbial ligands and to danger signals produced by the host during infection, and initiate downstream cascades that activate both innate and adaptive immunity. TLRs are expressed by professional immune cells and by the large majority of non-hematopoietic cells, including epithelial cells. In epithelial tissues, TLR functions are particularly important because these sites are constantly exposed to microorganisms, due to their location at the host interface with the environment. While at these sites specific defense mechanisms and inflammatory responses are initiated via TLR signaling against pathogens, suppression or lack of TLR activation is also observed in response to the commensal microbiota. The mechanisms by which TLR signaling is regulated in mucosal epithelial cells include differential expression and levels of TLRs (and their signaling partners), their cellular localization and positioning within the tissue in a fashion that favors responses to pathogens while dampening responses to commensals and maintaining tissue homeostasis in physiologic conditions. In this review, the expression and activation of TLRs in mucosal epithelial cells of several sites of the human body are examined. Specifically, the oral cavity, the ear canal and eye, the airways, the gut, and the reproductive tract are discussed, along with how site-specific host defense mechanisms are implemented via TLR signaling. PMID:25161655

  6. Rebamipide suppresses PolyI:C-stimulated cytokine production in human conjunctival epithelial cells.

    PubMed

    Ueta, Mayumi; Sotozono, Chie; Yokoi, Norihiko; Kinoshita, Shigeru

    2013-09-01

    We previously documented that ocular surface epithelial cells could regulate ocular surface inflammation and suggested that, while Toll-like receptor 3 upregulates, EP3, one of the prostaglandin E2 receptors, downregulates ocular surface inflammation. Others reported that rebamipide, a gastroprotective drug, could not only increase the gastric mucus production, but also suppressed gastric mucosal inflammation and that it was dominantly distributed in mucosal tissues. The eyedrop form of rebamipide, approved in Japan for use in the treatment of dry eye diseases, upregulates mucin secretion and production, thereby suppressing superficial punctate keratopathy on the ocular surface of patients with this disease. In the current study, we investigated whether rebamipide has anti- inflammatory effects on the ocular surface. To examine the effects of rebamipide on polyI:C-induced cytokine expression by primary human conjunctival epithelial cells, we used enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction assay. We studied the effects of rebamipide on ocular surface inflammation in our murine experimental allergic conjunctivitis (EAC) model. Rebamipide could suppress polyI:C-induced cytokine production and the expression of mRNAs for CXCL10, CXCL11, RANTES, MCP-1, and IL-6 in human conjunctival epithelial cells. In our EAC model, the topical administration of rebamipide suppressed conjunctival allergic eosinophil infiltration. The topical application of rebamipide on the ocular surface might suppress ocular surface inflammation by suppressing the production of cytokines by ocular surface epithelial cells.

  7. Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

    PubMed Central

    Wynford-Thomas, D; Bond, J A; Wyllie, F S; Burns, J S; Williams, E D; Jones, T; Sheer, D; Lemoine, N R

    1990-01-01

    To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types. Images PMID:1697930

  8. Cryopreservation and Recovery of Human Endometrial Epithelial Cells with High Viability, Purity, and Functional Fidelity

    PubMed Central

    Chen, Joseph C.; Hoffman, Jacquelyn R.; Arora, Ripla; Perrone, Lila A.; Gonzalez-Gomez, Christian J; Vo, Kim Chi; Laird, Diana J.; Irwin, Juan C.; Giudice, Linda C.

    2015-01-01

    Objective To develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eEC) retaining molecular and functional characteristics of endometrial epithelium in vivo. Design This is an in vitro study using human endometrial cells. Setting University research laboratory. Patients Endometrial biopsies were obtained from premenopausal women undergoing benign gynecological procedures. Interventions Primary eEC were cryopreserved in 1% fetal bovine serum (FBS)/10% dimethyl sulfoxide (DMSO) in Defined Keratinocyte Serum Free Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium. Main Outcome Measures Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity. Results eEC recovered after cryopreservation (n=5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared to eSF, recovered eEC displayed increased (P<0.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<0.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eEC secrete levels of cytokines and growth factors comparable to freshly cultured eEC. Recovered eEC can formed a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN. Conclusion We have developed a protocol for cryopreservation of eEC in which recovered cells after thawing demonstrate morphological, transcriptomic, and functional characteristics of human endometrial epithelium in vivo. PMID:26515378

  9. Constitutive and regulated secretion of secretory leukocyte proteinase inhibitor by human intestinal epithelial cells.

    PubMed

    Si-Tahar, M; Merlin, D; Sitaraman, S; Madara, J L

    2000-06-01

    Epithelial cells participate in immune regulation and mucosal integrity by generating a range of biologically active mediators. In the intestine, little is known about the potential endogenous anti-inflammatory molecules. Secretory leukocyte proteinase inhibitor (SLPI) is a major serine proteinase inhibitor, a potent antibiotic, and thus a potential anti-inflammatory molecule, although it is not known if it is secreted by intestinal epithelial cells. We show, by reverse-transcription polymerase chain reaction, the presence of SLPI messenger RNA in human model intestinal epithelial cell lines (Caco2-BBE, T84, and HT29-Cl.19A) and human jejunum and colon biopsy specimens. The polymerase chain reaction product was cloned and sequenced and is identical to that of SLPI isolated previously from the human parotid gland. As analyzed by enzyme-linked immunosorbent assay, the constitutive secretion of SLPI occurs in a markedly polarized manner toward the apical surface and is enhanced by inflammatory mediators including tumor necrosis factor alpha and interleukin 1beta (approximately 3.5-fold increase over control value). SLPI release is also stimulated by activation of protein kinase C isoenzymes, but not by activation of adenosine 3',5'-cyclic monophosphate- or Ca(2+)-regulated signaling molecules. SLPI protein is detectable in intestinal lavage fluids collected from normal adult humans. Recombinant SLPI attenuates digestive enzyme (trypsin)- or leukocyte proteinase (elastase)-induced permeability alteration of a model epithelia in a dose-dependent manner. Moreover, SLPI exhibits an antibacterial activity against at least one major intestinal pathogen, Salmonella typhimurium. In contrast, SLPI does not influence epithelial barrier integrity as assessed by transepithelial conductance measurements or electrogenic ion transport. These results establish that human intestinal epithelium expresses and apically secretes SLPI, a molecule that may significantly contribute to the

  10. Aluminium chloride promotes anchorage-independent growth in human mammary epithelial cells.

    PubMed

    Sappino, André-Pascal; Buser, Raphaële; Lesne, Laurence; Gimelli, Stefania; Béna, Frédérique; Belin, Dominique; Mandriota, Stefano J

    2012-03-01

    Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspirants, and in the range of those recently measured in the human breast, results in loss of contact inhibition and anchorage-independent growth. These effects were preceded by an increase of DNA synthesis, DNA double strand breaks (DSBs), and senescence in proliferating cultures. AlCl(3) also induced DSBs and senescence in proliferating primary human mammary epithelial cells. In contrast, it had no similar effects on human keratinocytes or fibroblasts, and was not detectably mutagenic in bacteria. MCF-10A cells morphologically transformed by long-term exposure to AlCl(3) display strong upregulation of the p53/p21(Waf1) pathway, a key mediator of growth arrest and senescence. These results suggest that aluminium is not generically mutagenic, but similar to an activated oncogene, it induces proliferation stress, DSBs and senescence in normal mammary epithelial cells; and that long-term exposure to AlCl(3) generates and selects for cells able to bypass p53/p21(Waf1) -mediated cellular senescence. Our observations do not formally identify aluminium as a breast carcinogen, but challenge the safety ascribed to its widespread use in underarm cosmetics. Copyright © 2012 John Wiley & Sons, Ltd.

  11. Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells

    PubMed Central

    Gotoh, Shimpei; Ito, Isao; Nagasaki, Tadao; Yamamoto, Yuki; Konishi, Satoshi; Korogi, Yohei; Matsumoto, Hisako; Muro, Shigeo; Hirai, Toyohiro; Funato, Michinori; Mae, Shin-Ichi; Toyoda, Taro; Sato-Otsubo, Aiko; Ogawa, Seishi; Osafune, Kenji; Mishima, Michiaki

    2014-01-01

    Summary No methods for isolating induced alveolar epithelial progenitor cells (AEPCs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs), we identified carboxypeptidase M (CPM) as a surface marker of NKX2-1+ “ventralized” anterior foregut endoderm cells (VAFECs) in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM+ cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM+ cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine. PMID:25241738

  12. Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

    PubMed Central

    Cabada, Miguel M.; Nichols, Joan; Gomez, Guillermo; White, A. Clinton

    2013-01-01

    The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens. PMID:23509153

  13. Regulation of membrane-associated mucins in the human corneal epithelial cells by dexamethasone.

    PubMed

    Seo, Kyoung Yul; Chung, So-Hyang; Lee, Joon H; Park, Mi Young; Kim, Eung Kweon

    2007-07-01

    To study the influence of dexamethasone on membrane-associated mucins produced by human corneal epithelial cells. Human corneal epithelial cells were cultured in medium supplemented with various concentrations of dexamethasone (ranging from 10 to 10 M). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis using monoclonal antibodies specific for human MUC1 (HMFG-1), MUC4 (1G8), and MUC16 (OC125) were performed to evaluate the effect of dexamethasone on membrane-associated mucin expression. The effect of glucocorticoid receptor antagonist (RU38486) on dexamethasone-induced mucin expression was estimated. RT-PCR revealed that MUC1 and MUC16 gene expression were upregulated 48 hours after addition of dexamethasone and that MUC4 gene expression was downregulated in the same condition. Western blot analysis showed that MUC1 and MUC16 proteins were increased after addition of dexamethasone. However, MUC4 was not detected by anti-MUC4 monoclonal antibody (1G8) for ASGP-2 under our conditions. Treatment with RU38486 inhibited the changes of MUC1, MUC4, and MUC16 by dexamethasone; thus, the effect of dexamethasone on mucin expression is mediated by glucocorticoid receptors. This study shows that MUC1, MUC4, and MUC16 are regulated differently by dexamethasone in human corneal epithelial cells. External application of dexamethasone might affect the precorneal mucin.

  14. Epigenetic influences of low-dose bisphenol A in primary human breast epithelial cells

    SciTech Connect

    Weng, Yu-I; Hsu, Pei-Yin; Liyanarachchi, Sandya

    Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ER{alpha} signaling pathway and global gene expressionmore » profiles. Compared to control cells, nuclear internalization of ER{alpha} was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ER{alpha}-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ER{alpha}-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.« less

  15. Neisseria gonorrhoeae infects the human endocervix by activating non-muscle myosin II-mediated epithelial exfoliation

    PubMed Central

    Yu, Qian; Lin, Brian; Qiu, Jessica; Stein, Daniel C.

    2017-01-01

    Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation. PMID:28406994

  16. Efficient generation of functional CFTR-expressing airway epithelial cells from human pluripotent stem cells.

    PubMed

    Wong, Amy P; Chin, Stephanie; Xia, Sunny; Garner, Jodi; Bear, Christine E; Rossant, Janet

    2015-03-01

    Airway epithelial cells are of great interest for research on lung development, regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation, and full maturation of the cells in air-liquid interface cultures occurs in <5 weeks. This protocol can be used for drug discovery, tissue regeneration or disease modeling.

  17. N-Myc Drives Neuroendocrine Prostate Cancer Initiated from Human Prostate Epithelial Cells.

    PubMed

    Lee, John K; Phillips, John W; Smith, Bryan A; Park, Jung Wook; Stoyanova, Tanya; McCaffrey, Erin F; Baertsch, Robert; Sokolov, Artem; Meyerowitz, Justin G; Mathis, Colleen; Cheng, Donghui; Stuart, Joshua M; Shokat, Kevan M; Gustafson, W Clay; Huang, Jiaoti; Witte, Owen N

    2016-04-11

    MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance, and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Gene Regulation by Retinoid Receptors in Human Mammary Epithelial Cells

    DTIC Science & Technology

    2002-10-01

    ceptors for collagen and fibronectin in human fibrosarcoma cells possessing Xia, Y-, S.G. Gil, and W.G. Carter. 1996. Anchorage mediated by integrin...simultaneously block all TdT terminal deoxynucleotidyl transferase pRB retinoblastoma protein wild-type RAR isoforms (-o, -P, and -- ) (Damm et CAT ...P0 pM treated with 0, 0,1, 1.0, or 10 jIM ATRA for 24 hours and cell lysates were assayed for CAT reporter activity. pCMV-GH was used as a transfection

  19. Effect of Estrogen on Mutagenesis in Human Mammary Epithelial Cells

    DTIC Science & Technology

    2005-06-01

    instability remains undefined in most human cancers, it appears to arise from subtle, intragenic mutations of the genes , whose products play a key role in...cells and is less labor-intensive. A G-G or T-G mismatch was introduced into ATG start codon of the enhanced green fluorescent protein (EGFP) gene ...Repair of the G-G or T-G mismatch to G-C or T-A, respectively in the heteroduplex plasmid generates a functional EGFP gene expression. The heteroduplex

  20. Organic electrochemical transistor array for recording transepithelial ion transport of human airway epithelial cells.

    PubMed

    Yao, Chunlei; Xie, Changyan; Lin, Peng; Yan, Feng; Huang, Pingbo; Hsing, I-Ming

    2013-12-03

    An organic electrochemical transistor array is integrated with human airway epithelial cells. This integration provides a novel method to couple transepithelial ion transport with electrical current. Activation and inhibition of transepithelial ion transport are readily detected with excellent time resolution. The organic electrochemical transistor array serves as a promising platform for physiological studies and drug testing. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Expression of a functional asialoglycoprotein receptor in human renal proximal tubular epithelial cells.

    PubMed

    Seow, Ying-ying T; Tan, Michelle G K; Woo, Keng Thye

    2002-07-01

    The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake. Copyright 2002 S. Karger AG, Basel

  2. Effect of resveratrol and zinc on intracellular zinc status in normal human prostate epithelial cells

    USDA-ARS?s Scientific Manuscript database

    To evaluate the influence of resveratrol on cellular zinc status, normal human prostate epithelial (NHPrE) cells were treated with 6 levels of resveratrol (0, 0.5, 1, 2.5, 5 and 10 microM) and 4 levels of zinc [0, 4, 16, and 32 microM for zinc-deficient (ZD), zinc-normal (ZN), zinc-adequate (ZA), an...

  3. Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

    PubMed

    Choo, C K; Ling, M T; Chan, K W; Tsao, S W; Zheng, Z; Zhang, D; Chan, L C; Wong, Y C

    1999-08-01

    The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis. Replication-defective retrovirus harboring the human papillomavirus (HPV) type 16 E6 and E7 open reading frames was used to infect primary human prostate epithelial cells. Polymerase chain reaction, followed by Southern hybridization for the HPV 16 E6/E7, Western blot for prostatic acid phosphatase, telomeric repeat amplification protocol assay for telomerase activity, two-dimensional gels for cytokeratins, and cytogenetic analysis were undertaken to characterized the infected cells. The retrovirus-infected cell line, HPr-1, continued to grow in culture for more than 80 successive passages. Normal primary cells failed to proliferate after passage 6. HPr-1 cells bore close resemblance to normal primary prostate epithelial cells, both morphologically and biochemically. However, they possessed telomerase activity and proliferated indefinitely. Cytogenetic analysis of HPr-1 cells revealed a human male karyotype with clonal abnormalities and the appearance of multiple double minutes. The HPr-1 cells expressed prostatic acid phosphatase and cytokeratins K8 and K18, proving that they were prostate epithelial cells. They were benign in nude mice tumor formation and soft agar colony formation assay. The HPr-1 cell line is an in vitro representation of early prostate neoplastic progression. Copyright 1999 Wiley-Liss, Inc.

  4. IL-17 suppresses immune effector functions in human papillomavirus-associated epithelial hyperplasia.

    PubMed

    Gosmann, Christina; Mattarollo, Stephen R; Bridge, Jennifer A; Frazer, Ian H; Blumenthal, Antje

    2014-09-01

    Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia that can progress to cancer and is thought to depend on immunosuppressive mechanisms that prevent viral clearance by the host. IL-17 is a cytokine with diverse functions in host defense and in the pathology of autoimmune disorders, chronic inflammatory diseases, and cancer. We analyzed biopsies from patients with HPV-associated cervical intraepithelial neoplasia grade 2/3 and murine skin displaying HPV16 E7 protein-induced epithelial hyperplasia, which closely models hyperplasia in chronic HPV lesions. Expression of IL-17 and IL-23, a major inducer of IL-17, was elevated in both human HPV-infected and murine E7-expressing lesions. Using a skin-grafting model, we demonstrated that IL-17 in HPV16 E7 transgenic skin grafts inhibited effective host immune responses against the graft. IL-17 was produced by CD3(+) T cells, predominantly CD4(+) T cells in human, and CD4(+) and γδ T cells in mouse hyperplastic lesions. IL-23 and IL-1β, but not IL-18, induced IL-17 production in E7 transgenic skin. Together, these findings demonstrate an immunosuppressive role for IL-17 in HPV-associated epithelial hyperplasia and suggest that blocking IL-17 in persistent viral infection may promote antiviral immunity and prevent progression to cancer. Copyright © 2014 by The American Association of Immunologists, Inc.

  5. 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

  6. Induction of proinflammatory cytokines in human lung epithelial cells during Rhodococcus equi infection.

    PubMed

    Remuzgo-Martínez, Sara; Pilares-Ortega, Lilian; Alvarez-Rodríguez, Lorena; Aranzamendi-Zaldunbide, Maitane; Padilla, Daniel; Icardo, Jose Manuel; Ramos-Vivas, Jose

    2013-08-01

    Rhodococcus equi is an opportunistic human pathogen associated with immunosuppressed people. While the interaction of R. equi with macrophages has been comprehensively studied, little is known about its interactions with non-phagocytic cells. Here, we characterized the entry process of this bacterium into human lung epithelial cells. The invasion is inhibited by nocodazole and wortmannin, suggesting that the phosphatidylinositol 3-kinase pathway and microtubule cytoskeleton are important for invasion. Pre-incubation of R. equi with a rabbit anti-R. equi polyclonal antiserum resulted in a dramatic reduction in invasion. Also, the invasion process as studied by immunofluorescence and scanning electron microscopy indicates that R. equi make initial contact with the microvilli of the A549 cells, and at the structural level, the entry process was observed to occur via a zipper-like mechanism. Infected lung epithelial cells upregulate the expression of cytokines IL-8 and IL-6 upon infection. The production of these pro-inflammatory cytokines was significantly enhanced in culture supernatants from cells infected with non-mucoid plasmid-less strains when compared with cells infected with mucoid strains. These results demonstrate that human airway epithelial cells produce pro-inflammatory mediators against R. equi isolates.

  7. Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

    PubMed

    Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

    2017-08-01

    An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    PubMed Central

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

    1982-01-01

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

  9. Effect of Lunar Dust Simulant on Human Epithelial Cell Lines

    NASA Technical Reports Server (NTRS)

    Myers, Nicholas J.; Wallace, William T.; Jeevarajan, Antony S.

    2009-01-01

    The purpose of this project is to assess the potential toxicity of lunar dust to cause the release of pro-inflammatory cytokines by human lung cells. Some of this dust is on the scale of 1-2 micrometers and could enter the lungs when astronauts track dust into the habitat and inhale it. This could be a serious problem as NASA plans on going back to the moon for an extended period of time. Literature shows that quartz, which has a known cytoxicity, can cause acute cases of silicosis within 6 months, and in most cases cause silicosis after 3 years. The activation of lunar dust through impacts creates surface based radicals which, upon contact with water create hydroxl radicals and peroxyl radicals which are very reactive and potentially might even be as cytotoxic as quartz. These radicals could then react with lung cells to produce pro-inflammatory mediators such as interleukin-6 and interleukin-8, and TNF-alpha.

  10. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  11. ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO UTAH VALLEY PARTICULATE MATTER

    EPA Science Inventory

    Exposure to ambient particulate matter (PM) in the Utah Valley (UV) has previously been associated with a variety of adverse health effects. To investigate intracellular signaling mechanisms for pulmonary responses to UV PM inhalation, human primary airway epithelial cells (NHBE)...

  12. IDENTIFICATION AND CHARACTERIZATION OF HUMAN AIRWAY EPITHELIAL CELL PROTEINS PHOSPHORYLATED IN RESPONSE TO PARTICULATE MATTER (PM) EXPOSURE.

    EPA Science Inventory

    Multiple studies conducted by NHEERL scientists in recent years have shown that acute exposure to metals found associated with combustion-derived particulate matter (PM) alters phosphoprotein metabolism in human airway epithelial cells causing intracellular signaling. This disreg...

  13. Complex epithelial remodeling underlie the fusion event in early fetal development of the human penile urethra.

    PubMed

    Shen, Joel; Overland, Maya; Sinclair, Adriane; Cao, Mei; Yue, Xuan; Cunha, Gerald; Baskin, Laurence

    We recently described a two-step process of urethral plate canalization and urethral fold fusion to form the human penile urethra. Canalization ("opening zipper") opens the solid urethral plate into a groove, and fusion ("closing zipper") closes the urethral groove to form the penile urethra. We hypothesize that failure of canalization and/or fusion during human urethral formation can lead to hypospadias. Herein, we use scanning electron microscopy (SEM) and analysis of transverse serial sections to better characterize development of the human fetal penile urethra as contrasted to the development of the human fetal clitoris. Eighteen 7-13 week human fetal external genitalia specimens were analyzed by SEM, and fifteen additional human fetal specimens were sectioned for histologic analysis. SEM images demonstrate canalization of the urethral/vestibular plate in the developing male and female external genitalia, respectively, followed by proximal to distal fusion of the urethral folds in males only. The fusion process during penile development occurs sequentially in multiple layers and through the interlacing of epidermal "cords". Complex epithelial organization is also noted at the site of active canalization. The demarcation between the epidermis of the shaft and the glans becomes distinct during development, and the epithelial tag at the distal tip of the penile and clitoral glans regresses as development progresses. In summary, SEM analysis of human fetal specimens supports the two-zipper hypothesis of formation of the penile urethra. The opening zipper progresses from proximal to distal along the shaft of the penis and clitoris into the glans in identical fashion in both sexes. The closing zipper mechanism is active only in males and is not a single process but rather a series of layered fusion events, uniquely different from the simple fusion of two epithelial surfaces as occurs in formation of the palate and neural tube. Copyright © 2016 International Society

  14. Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

    PubMed Central

    Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

    2013-01-01

    Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

  15. Bladder surface glycosaminoglycans is a human epithelial permeability barrier.

    PubMed

    Lilly, J D; Parsons, C L

    1990-12-01

    Transitional epithelium of the bladder has been known to be impermeable. The data reported herein suggest the principal barrier to permeability may be glycosaminoglycans (GAG) of the surface of the bladder. We examined the ability of surface GAG to prevent a small molecule, urea, from moving across the epithelium in humans. It appears that GAG provide a physical barrier which prevents small molecules from reaching the underlying tight junctions and cell membranes and, hence, are a major permeability barrier. Normal volunteers (27) had 100 milliliters of a 200 grams per liter urea solution placed into their bladders for 45 minutes. Net flow of urea from the bladder lumen was 5.1 per cent. Volunteers who were capable of completing the study (19) had protamine sulfate (5 milligrams per milliliter) instilled in the bladder for 15 minutes, then removed and a second urea study done. Urea loss was significantly higher at 22 per cent (p less than 0.02). A solution of heparin (2,000 units per milliliter) was instilled for 15 minutes followed by a third urea study and urea loss was reversed to 9 per cent. All volunteers experienced significant urinary urgency and discomfort after protamine treatment which were reduced by heparin.

  16. Yersinia enterocolitica-Induced Interleukin-8 Secretion by Human Intestinal Epithelial Cells Depends on Cell Differentiation

    PubMed Central

    Schulte, Ralf; Autenrieth, Ingo B.

    1998-01-01

    In response to bacterial entry epithelial cells up-regulate expression and secretion of various proinflammatory cytokines, including interleukin-8 (IL-8). We studied Yersinia enterocolitica O:8-induced IL-8 secretion by intestinal epithelial cells as a function of cell differentiation. For this purpose, human T84 intestinal epithelial cells were grown on permeable supports, which led to the formation of tight monolayers of polarized intestinal epithelial cells. To analyze IL-8 secretion as a function of cell differentiation, T84 monolayers were infected from the apical or basolateral side at different stages of differentiation. Both virulent (plasmid-carrying) and nonvirulent (plasmid-cured) Y. enterocolitica strains invaded nondifferentiated T84 cells from the apical side. Yersinia invasion into T84 cells was followed by secretion of IL-8. After polarized differentiation of T84 cells Y. enterocolitica was no longer able to invade from the apical side or to induce IL-8 secretion by T84 cells. However, Y. enterocolitica invaded and induced IL-8 secretion by polarized T84 cells after infection from the basolateral side. Basolateral invasion required the presence of the Yersinia invasion locus, inv, suggesting β1 integrin-mediated cell invasion. After basolateral infection, Yersinia-induced IL-8 secretion was not strictly dependent on cell invasion. Thus, although the plasmid-carrying Y. enterocolitica strain did not significantly invade T84 cells, it induced significant IL-8 secretion. Taken together, these data show that Yersinia-triggered IL-8 secretion by intestinal epithelial cells depends on cell differentiation and might be induced by invasion as well as by basolateral adhesion, suggesting that invasion is not essential for triggering IL-8 production. Whether IL-8 secretion is involved in the pathogenesis of Yersinia-induced abscess formation in Peyer’s patch tissue remains to be shown. PMID:9488416

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

    PubMed Central

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

    2013-01-01

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

  18. CD147 Promotes Entry of Pentamer-Expressing Human Cytomegalovirus into Epithelial and Endothelial Cells

    PubMed Central

    Pritchard, Sarah R.; Wisner, Todd W.; Liu, Jing; Jardetzky, Ted S.; Johnson, David C.

    2018-01-01

    ABSTRACT Human cytomegalovirus (HCMV) replicates in many diverse cell types in vivo, and entry into different cells involves distinct entry mechanisms and different envelope glycoproteins. HCMV glycoprotein gB is thought to act as the virus fusogen, apparently after being triggered by different gH/gL proteins that bind distinct cellular receptors or entry mediators. A trimer of gH/gL/gO is required for entry into all cell types, and entry into fibroblasts involves trimer binding to platelet-derived growth factor receptor alpha (PDGFRα). HCMV entry into biologically relevant epithelial and endothelial cells and monocyte-macrophages also requires a pentamer, gH/gL complexed with UL128, UL130, and UL131, and there is evidence that the pentamer binds unidentified receptors. We screened an epithelial cell cDNA library and identified the cell surface protein CD147, which increased entry of pentamer-expressing HCMV into HeLa cells but not entry of HCMV that lacked the pentamer. A panel of CD147-specific monoclonal antibodies inhibited HCMV entry into epithelial and endothelial cells, but not entry into fibroblasts. shRNA silencing of CD147 in endothelial cells inhibited HCMV entry but not entry into fibroblasts. CD147 colocalized with HCMV particles on cell surfaces and in endosomes. CD147 also promoted cell-cell fusion induced by expression of pentamer and gB in epithelial cells. However, soluble CD147 did not block HCMV entry and trimer and pentamer did not bind directly to CD147, supporting the hypothesis that CD147 acts indirectly through other proteins. CD147 represents the first HCMV entry mediator that specifically functions to promote entry of pentamer-expressing HCMV into epithelial and endothelial cells. PMID:29739904

  19. Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma.

    PubMed

    Looi, K; Buckley, A G; Rigby, P J; Garratt, L W; Iosifidis, T; Zosky, G R; Larcombe, A N; Lannigan, F J; Ling, K-M; Martinovich, K M; Kicic-Starcevich, E; Shaw, N C; Sutanto, E N; Knight, D A; Kicic, A; Stick, S M

    2018-05-01

    Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; R T ) and permeability to fluorescent dextran. Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV

  20. Development of epithelial and mesenchymal regionalization of the human fetal utero-vaginal anlagen

    PubMed Central

    Fritsch, Helga; Hoermann, Romed; Bitsche, Mario; Pechriggl, Elisabeth; Reich, Olaf

    2013-01-01

    Literature on the development of the human vagina is abundant; however, contributions concerning the prenatal development of the entire utero-vaginal anlagen (UVA) are rare or carried out in rodents. The primary epithelial characteristics in the adult vagina and uterus are determined during prenatal development and depend on epithelio-mesenchymal stroma interaction; thus an investigation summarizing the spatiotemporal distribution of relevant molecular markers in the entire human UVA will be of current interest. We phenotyped epithelial and mesenchymal characteristics in sagittal sections from 24 female fetuses of 14–34 weeks of gestation and two female newborns by immunostaining with cytokeratins 8, 13, 14 and 17, p63, bcl-2, bmp4, HOX A13, CD31, VEGF, SMA, Pax2 and vimentin. Epithelial differentiation followed a caudal-to-cranial direction in the UVA. Due to the cytokeratin profile of cytokeratins 8, 13 and 14, the characteristics of the different epithelial zones in the UVA could already be recognized in middle-age fetuses. Vaginal epithelium originated from the urogenital sinus in the lower portion and initiated the transformation of vimentin-positive Müllerian epithelium in the upper vaginal portion. During prenatal development the original squamo-columnar junction was clearly detectable from week 24 onwards and was always found in the cervical canal. Early blc-2 positivity within the surrounding mesenchyme of the entire vagina including the portio region pointed to an organ-specific mesenchymal influence. Prenatal findings in human specimens clearly show that fornix epithelium up to the squamo-columnar junction is of vaginal Müllerian origin, and the cervical epithelium cranial to the squamo-columnar junction is of uterine Müllerian origin and includes cells with enough plasticity to transform into squamous epithelium. PMID:23406280

  1. Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells.

    PubMed

    Wu, Qun; Jiang, Di; Minor, Maisha; Chu, Hong Wei

    2014-01-01

    The use of electronic cigarettes (e-cigarettes) is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV) infection. We examined the effects of e-cigarette liquid (e-liquid) on pro-inflammatory cytokine (e.g., IL-6) production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1) in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes.

  2. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.

    PubMed

    Sato, Toshiro; Stange, Daniel E; Ferrante, Marc; Vries, Robert G J; Van Es, Johan H; Van den Brink, Stieneke; Van Houdt, Winan J; Pronk, Apollo; Van Gorp, Joost; Siersema, Peter D; Clevers, Hans

    2011-11-01

    We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  3. The parietal epithelial cell is crucially involved in human idiopathic focal segmental glomerulosclerosis.

    PubMed

    Dijkman, Henry; Smeets, Bart; van der Laak, Jeroen; Steenbergen, Eric; Wetzels, Jack

    2005-10-01

    Focal segmental glomerulosclerosis (FSGS) is one of the most common patterns of glomerular injury encountered in human renal biopsies. Epithelial hyperplasia, which can be prominent in FSGS, has been attributed to dedifferentiation and proliferation of podocytes. Based on observations in a mouse model of FSGS, we pointed to the role of parietal epithelial cells (PECs). In the present study we investigated the relative role of PECs and podocytes in human idiopathic FSGS. We performed a detailed study of lesions from a patient with recurrent idiopathic FSGS by serial sectioning, marker analysis and three-dimensional reconstruction of glomeruli. We have studied the expression of markers for podocytes, PECs, mesangial cells, endothelium, and myofibroblasts. We also looked at proliferation and composition of the deposited extracellular matrix (ECM). We found that proliferating epithelial cells in FSGS lesions are negative for podocyte and macrophage markers, but stain for PEC markers. The composition of the matrix deposited by these cells is identical to Bowman's capsule. Our study demonstrates that PECs are crucially involved in the pathogenesis of FSGS lesions.

  4. Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

    PubMed

    Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

    2012-12-01

    The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

  5. Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids.

    PubMed

    Hill, David R; Huang, Sha; Tsai, Yu-Hwai; Spence, Jason R; Young, Vincent B

    2017-12-18

    Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

  6. Inflammatory signaling pathways induced by Helicobacter pylori in primary human gastric epithelial cells.

    PubMed

    Tran, Cong Tri; Garcia, Magali; Garnier, Martine; Burucoa, Christophe; Bodet, Charles

    2017-02-01

    Inflammatory signaling pathways induced by Helicobacter pylori remain unclear, having been studied mostly on cell-line models derived from gastric adenocarcinoma with potentially altered signaling pathways and nonfunctional receptors. Here, H. pylori-induced signaling pathways were investigated in primary human gastric epithelial cells. Inflammatory response was analyzed on chemokine mRNA expression and production after infection of gastric epithelial cells by H. pylori strains, B128 and B128Δ cagM, a cag type IV secretion system defective strain. Signaling pathway involvement was investigated using inhibitors of epidermal growth factor receptor (EGFR), MAPK, JAK and blocking Abs against TLR2 and TLR4. Inhibitors of EGFR, MAPK and JAK significantly reduced the chemokine mRNA expression and production induced by both H. pylori strains at 3 h and 24 h post-infection. JNK inhibitor reduced chemokine production at 24 h post-infection. Blocking Abs against TLR2 but not TLR4 showed significant reduction of chemokine secretion. Using primary culture of human gastric epithelial cells, our data suggest that H. pylori can be recognized by TLR2, leading to chemokine induction, and that EGFR, MAPK and the JAK/STAT signaling pathways play a key role in the H. pylori-induced CXCL1, CXCL5 and CXCL8 response in a cag pathogenicity island-independent manner.

  7. Oligosaccharide receptor mimics inhibit Legionella pneumophila attachment to human respiratory epithelial cells.

    PubMed

    Thomas, Richard J; Brooks, Tim J

    2004-02-01

    Legionnaire's disease is caused by the intracellular pathogen Legionella pneumophila, presenting as an acute pneumonia. Attachment is the key step during infection, often relying on an interaction between host cell oligosaccharides and bacterial adhesins. Inhibition of this interaction by receptor mimics offers possible novel therapeutic treatments. L. pneumophila attachment to the A549 cell line was significantly reduced by treatment with tunicamycin (73.6%) and sodium metaperiodate (63.7%). This indicates the importance of cell surface oligosaccharide chains in adhesion. A number of putative anti-adhesion compounds inhibited attachment to the A549 and U937 cell lines. The most inhibitory compounds were polymeric saccharides, GalNAcbeta1-4Gal, Galbeta1-4GlcNAc and para-nitrophenol. These compounds inhibited adhesion to a range of human respiratory cell lines, including nasal epithelial, bronchial epithelial and alveolar epithelial cell lines and the human monocytic cell line, U937. Some eukaryotic receptors for L. pneumophila were determined to be the glycolipids, asialo-GM1 and asialo-GM2 that contain the inhibitory saccharide moiety, GalNAcbeta1-4Gal. The identified compounds have the potential to be used as novel treatments for Legionnaire's disease.

  8. IL-29 Enhances CXCL10 Production in TNF-α-stimulated Human Oral Epithelial Cells.

    PubMed

    Hosokawa, Yoshitaka; Hosokawa, Ikuko; Shindo, Satoru; Ozaki, Kazumi; Matsuo, Takashi

    2017-08-01

    Interleukin-29 (IL-29) is a cytokine belonging to the Type III interferon family. It was recently detected in the gingival crevicular fluid of periodontitis patients. However, the role of IL-29 in the pathogenesis of periodontal disease remains unknown. The aim of this study was to examine the effects of IL-29 on C-X-C motif chemokine ligand 10 (CXCL10) production in human oral epithelial cells. We measured CXCL10 production in TR146 cells, which is a human oral epithelial cell line, using an enzyme-linked immunosorbent assay. We used a Western blot analysis to detect IL-29 receptor expression and the phosphorylation levels of signal transduction molecules, including p38 mitogen-activated protein kinases (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor (NF)- κB p65, in the TR146 cells. The TR146 cells expressed the IL-29 receptor. IL-29 induced CXCL10 production in the TR146 cells. IL-29 significantly enhanced CXCL10 production in tumor necrosis factor (TNF)-α-stimulated TR146 cells. The p38 MAPK, STAT3, and NF-κB pathways were found to be related to the IL-29-induced enhancement of CXCL10 production in TNF-α-stimulated TR146 cells. IL-29 promotes T helper 1-cell accumulation in periodontal lesions by inducing CXCL10 production in oral epithelial cells.

  9. Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells.

    PubMed Central

    Kelley, T J; Drumm, M L

    1998-01-01

    It has been reported that exhaled nitric oxide levels are reduced in cystic fibrosis (CF) patients. We have examined the inducible isoform of nitric oxide synthase (iNOS) in the airways by immunostaining and found that iNOS is constitutively expressed in the airway epithelia of non-CF mouse and human tissues but essentially absent in the epithelium of CF airways. We explored potential consequences of lost iNOS expression and found that iNOS inhibition significantly increases mouse nasal trans-epithelial potential difference, and hindered the ability of excised mouse lungs to prevent growth of Pseudomonas aeruginosa. The absence of continuous nitric oxide production in epithelial cells of CF airways may play a role in two CF-associated characteristics: hyperabsorption of sodium and susceptibility to bacterial infections. PMID:9739054

  10. Butyrate modulating effects on pro-inflammatory pathways in human intestinal epithelial cells.

    PubMed

    Elce, A; Amato, F; Zarrilli, F; Calignano, A; Troncone, R; Castaldo, G; Canani, R B

    2017-10-13

    Butyrate acts as energy source for intestinal epithelial cells and as key mediator of several immune processes, modulating gene expression mainly through histone deacetylation inhibition. Thanks to these effects, butyrate has been proposed for the treatment of many intestinal diseases. Aim of this study was to investigate the effect of butyrate on the expression of a large series of target genes encoding proteins involved in pro-inflammatory pathways. We performed quantitative real-time-PCR analysis of the expression of 86 genes encoding proteins bearing to pro-inflammatory pathways, before and after butyrate exposure, in primary epithelial cells derived from human small intestine and colon. Butyrate significantly down-regulated the expression of genes involved in inflammatory response, among which nuclear factor kappa beta, interferon-gamma, Toll like 2 receptor and tumour necrosis factor-alpha. Further confirmations of these data, including studies at protein level, would support the use of butyrate as effective therapeutic strategy in intestinal inflammatory disorders.

  11. Generation of Epithelial Cell Populations from Human Pluripotent Stem Cells Using a Small-Molecule Inhibitor of Src Family Kinases.

    PubMed

    Selekman, Joshua A; Lian, Xiaojun; Palecek, Sean P

    2016-01-01

    Human pluripotent stem cells (hPSCs), under the right conditions, can be engineered to generate populations of any somatic cell type. Knowledge of what mechanisms govern differentiation towards a particular lineage is often quite useful for efficiently producing somatic cell populations from hPSCs. Here, we have outlined a strategy for deriving populations of simple epithelial cells, as well as more mature epidermal keratinocyte progenitors, from hPSCs by exploiting a mechanism previously shown to direct epithelial differentiation of hPSCs. Specifically, we describe how to direct epithelial differentiation of hPSCs using an Src family kinase inhibitor, SU6656, which has been shown to modulate β-catenin translocation to the cell membrane and thus promote epithelial differentiation. The differentiation platform outlined here produces cells with the ability to terminally differentiate to epidermal keratinocytes in culture through a stable simple epithelial cell intermediate that can be expanded in culture for numerous (>10) passages.

  12. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    PubMed

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley

  13. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Yang, Ruifeng; Zheng, Ying; Burrows, Michelle; Liu, Shujing; Wei, Zhi; Nace, Arben; Guo, Wei; Kumar, Suresh; Cotsarelis, George; Xu, Xiaowei

    2014-01-01

    Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair-specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

  14. Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro.

    PubMed

    Cruickshank, S M; Southgate, J; Selby, P J; Trejdosiewicz, L K

    1998-10-01

    Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

  15. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    NASA Technical Reports Server (NTRS)

    Goodwin, T. J.; McCarthy, M.; Lin, Y-H

    2006-01-01

    In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

  16. Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

    PubMed

    Gingerich, Aaron; Pang, Lan; Hanson, Jarod; Dlugolenski, Daniel; Streich, Rebecca; Lafontaine, Eric R; Nagy, Tamás; Tripp, Ralph A; Rada, Balázs

    2016-01-01

    Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

  17. In vitro effects of three blood derivatives on human corneal epithelial cells.

    PubMed

    Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Durán, Juan A; Morales, María-Celia

    2012-08-15

    We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

  18. Three-dimensional epithelial tissues generated from human embryonic stem cells.

    PubMed

    Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

    2009-11-01

    The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

  19. SERS and integrative imaging upon internalization of quantum dots into human oral epithelial cells.

    PubMed

    Cepeda-Pérez, Elisa; López-Luke, Tzarara; Plascencia-Villa, Germán; Perez-Mayen, Leonardo; Ceja-Fdez, Andrea; Ponce, Arturo; Vivero-Escoto, Juan; de la Rosa, Elder

    2016-07-01

    CdTe quantum dots (QDs) are widely used in bio-applications due to their size and highly efficient optical properties. However internalization mechanisms thereof for the variety of freshly extracted, not cultivated human cells and their specific molecular interactions remains an open topic for discussion. In this study, we assess the internalization mechanism of CdTe quantum dots (3.3 nm) capped with thioglycolic acid using non cultivated oral epithelial cells obtained from healthy donors. Naked gold nanoparticles (40 nm) were successfully used as nanosensors for surface-enhanced Raman spectroscopy to efficiently identify characteristic Raman peaks, providing new evidence indicating that the first interactions of these QDs with epithelial cells occurred preferentially with aromatic rings and amine groups of amino acid residues and glycans from trans-membrane proteins and cytoskeleton. Using an integrative combination of advanced imaging techniques, including ultra-high resolution SEM, high resolution STEM coupled with EDX spectroscopy together with the results obtained by Raman spectroscopy, it was determined that thioglycolic acid capped CdTe QDs are efficiently internalized into freshly extracted oral epithelial cells only by facilitated diffusion, distributed into cytoplasm and even within the cell nucleus in three minutes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. AntogomiR-451 protects human gastric epithelial cells from ethanol via activating AMPK signaling.

    PubMed

    Zhu, Huanhuan; Zhang, Linjie; Xu, Jianmin; Zhu, Chunhua; Zhao, Hui; Zhu, Yongkang; Lv, Guoqiang

    2018-02-26

    The prevention and treatment efficiency of ethanol-induced gastric epithelial injury are not satisfied. We have previously shown that AMP-activated protein kinase (AMPK) activation exerts a pro-survival function in human gastric epithelial cells (GECs). miroRNA-451 ("miR-451")'s inhibitor, antagomiR-451, can activate AMPK signaling. In the present study, we show that forced-expression of antagomiR-451 via a lentiviral vector depleted miR-451, leading to AMPK activation in established GES-1 cells and primary human GECs. AntagomiR-451 efficiently protected GES-1 cells and primary human GECs from ethanol-induced viability reduction and apoptosis. AMPK activation is required for antagomiR-451-induced GEC protection. AMPKα1 knockdown (by targeted-shRNAs) or knockout (by CRISPR-Cas-9 KO plasmid) blocked antagomiR-451-induced AMPK activation, and GEC protection against ethanol. Further experimental results show that antagomiR-451 significantly attenuated ethanol-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage. Collectively, antagomiR-451 protects human GECs from ethanol via activating AMPK signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion.

    PubMed

    Cuman, Carly; Van Sinderen, Michelle; Gantier, Michael P; Rainczuk, Kate; Sorby, Kelli; Rombauts, Luk; Osianlis, Tiki; Dimitriadis, Evdokia

    2015-10-01

    Successful embryo implantation requires synchronous development and communication between the blastocyst and the endometrium, however the mechanisms of communication in humans are virtually unknown. Recent studies have revealed that microRNAs (miRs) are present in bodily fluids and secreted by cells in culture. We have identified that human blastocysts differentially secrete miRs in a pattern associated with their implantation outcome. miR-661 was the most highly expressed miR in blastocyst culture media (BCM) from blastocysts that failed to implant (non-implanted) compared to blastocysts that implanted (implanted). Our results indicate a possible role for Argonaute 1 in the transport of miR-661 in non-implanted BCM and taken up by primary human endometrial epithelial cells (HEECs). miR-661 uptake by HEEC reduced trophoblast cell line spheroid attachment to HEEC via PVRL1. Our results suggest that human blastocysts alter the endometrial epithelial adhesion, the initiating event of implantation, via the secretion of miR, abnormalities in which result in implantation failure.

  2. Oxidative stress in Nipah virus-infected human small airway epithelial cells.

    PubMed

    Escaffre, Olivier; Halliday, Hailey; Borisevich, Viktoriya; Casola, Antonella; Rockx, Barry

    2015-10-01

    Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development.

  3. Silver nanowire interactions with primary human alveolar type-II epithelial cell secretions: contrasting bioreactivity with human alveolar type-I and type-II epithelial cells

    PubMed Central

    Sweeney, Sinbad; Theodorou, Ioannis G.; Zambianchi, Martina; Chen, Shu; Gow, Andrew; Schwander, Stephan; Zhang, Junfeng (Jim); Chung, Kian Fan; Shaffer, Milo S.; Ryan, Mary P.; Porter, Alexandra E.; Tetley, Teresa D.

    2015-01-01

    Inhaled nanoparticles have a high deposition rate in the alveolar units of the deep lung. The alveolar epithelium is composed of type-I and type-II epithelial cells (ATI and ATII respectively) and is bathed in pulmonary surfactant. The effect of native human ATII cell secretions on nanoparticle toxicity is not known. We investigated the cellular uptake and toxicity of silver nanowires (AgNWs; 70 nm diameter, 1.5 μm length) with human ATI-like cells (TT1), in the absence or presence of Curosurf® (a natural porcine pulmonary surfactant with a low amount of protein) or harvested primary human ATII cell secretions (HAS; containing both the complete lipid as well as the full protein complement of human pulmonary surfactant i.e. SP-A, SP-B, SP-C and SP-D). We hypothesised that Curosurf® or HAS would confer improved protection for TT1 cells, limiting the toxicity of AgNWs. In agreement with our hypothesis, HAS reduced the inflammatory and reactive oxygen species (ROS)-generating potential of AgNWs with exposed TT1 cells. For example, IL-8 release and ROS generation was reduced by 38% and 29%, respectively, resulting in similar levels to that of the non-treated controls. However in contrast to our hypothesis, Curosurf® had no effect. We found a significant reduction in AgNW uptake by TT1 cells in the presence of HAS but not Curosurf. Furthermore, we show that the SP-A and SP-D are likely to be involved in this process as they were found to be specifically bound to the AgNWs. While ATI cells appear to be protected by HAS, evidence suggested that ATII cells, despite no uptake, were vulnerable to AgNW exposure (indicated by increased IL-8 release and ROS generation and decreased intracellular SP-A levels one day post-exposure). This study provides unique findings that may be important for the study of lung epithelial-endothelial translocation of nanoparticles in general and associated toxicity within the alveolar unit. PMID:25996248

  4. Human lactoferrin stimulates skin keratinocyte function and wound re-epithelialization.

    PubMed

    Tang, L; Wu, J J; Ma, Q; Cui, T; Andreopoulos, F M; Gil, J; Valdes, J; Davis, S C; Li, J

    2010-07-01

    Human lactoferrin (hLF), a member of the transferrin family, is known for its antimicrobial and anti-inflammatory effects. Recent studies on various nonskin cell lines indicate that hLF may have a stimulatory effect on cell proliferation. To study the potential role of hLF in wound re-epithelialization. The effects of hLF on cell growth, migration, attachment and survival were assessed, with a rice-derived recombinant hLF (holo-rhLF), using proliferation analysis, scratch migration assay, calcein-AM/propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method, respectively. The mechanisms of hLF on cell proliferation and migration were explored using specific pathway inhibitors. The involvement of lactoferrin receptor low-density lipoprotein receptor-related protein 1 (LRP1) was examined with RNA interference technique. An in vivo swine second-degree burn wound model was also used to assess wound re-epithelialization. Studies revealed that holo-rhLF significantly stimulated keratinocyte proliferation which could be blocked by mitogen-activated protein kinase (MAPK) kinase 1 inhibitor. Holo-rhLF also showed strong promoting effects on keratinocyte migration, which could be blocked by either inhibition of the MAPK, Src and Rho/ROCK pathways, or downregulation of the LRP1 receptor. With cells under starving or 12-O-tetradecanoylphorbol-13-acetate exposure, the addition of holo-rhLF was found greatly to increase cell viability and inhibit cell apoptosis. Additionally, holo-rhLF significantly increased the rate of wound re-epithelialization in swine second-degree burn wounds. Our studies demonstrate the direct effects of holo-rhLF on wound re-epithelialization including the enhancement of keratinocyte proliferation and migration as well as the protection of cells from apoptosis. The data strongly indicate its potential therapeutic applications in wound healing.

  5. Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

    PubMed Central

    2013-01-01

    Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA

  6. Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells

    NASA Technical Reports Server (NTRS)

    Durante, M.; Grossi, G. F.; Gialanella, G.; Pugliese, M.; Nappo, M.; Yang, T. C.

    1995-01-01

    We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS).

  7. Establishment and characterization of a telomerase immortalized human gingival epithelial cell line.

    PubMed

    Moffatt-Jauregui, C E; Robinson, B; de Moya, A V; Brockman, R D; Roman, A V; Cash, M N; Culp, D J; Lamont, R J

    2013-12-01

    Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase immortalized human gingival epithelial cell line and compare its in vitro behaviour to that of human GECs. Human primary GECs were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, telomerase immortalized gingival keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors and invasion by P. gingivalis. TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for toll-like receptors 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild-type P. gingivalis and an invasion defective ΔserB mutant. Results confirm bmi1/hTERT immortalization of primary GECs generated a robust cell line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells. © 2013 John Wiley & Sons A/S. Published by John Wiley

  8. Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

    PubMed

    Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

    2018-02-20

    Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

  9. Regulation of Proinflammatory Cytokines in Human Lung Epithelial Cells Infected with Mycoplasma pneumoniae

    PubMed Central

    Yang, Jun; Hooper, W. Craig; Phillips, Donald J.; Talkington, Deborah F.

    2002-01-01

    Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1β mRNA also increased after infection and IL-1β protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level. PMID:12065506

  10. Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells.

    PubMed

    Durante, M; Grossi, G F; Gialanella, G; Pugliese, M; Nappo, M; Yang, T C

    1995-08-01

    We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

    SciTech Connect

    Huang, Chunrong; Zheng, Haichong; He, Wanmei

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activatedmore » the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.« less

  12. Apelin attenuates TGF-β1-induced epithelial to mesenchymal transition via activation of PKC-ε in human renal tubular epithelial cells.

    PubMed

    Wang, Li-Yan; Diao, Zong-Li; Zheng, Jun-Fang; Wu, Yi-Ru; Zhang, Qi-Dong; Liu, Wen-Hu

    2017-10-01

    Epithelial to mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of renal fibrosis. Apelin, a bioactive peptide, has recently been recognized to protect against renal profibrotic activity, but the underlying mechanism has not yet been elucidated. In this study, we investigated the regulation of EMT in the presence of apelin-13 in vitro. Expression of the mesenchymal marker alpha-smooth muscle actin (α-SMA) and the epithelial marker E-cadherin was examined by immunofluorescence and western blotting in transforming growth factor beta 1 (TGF-β1)-stimulated human proximal tubular epithelial cells. Expression of extracellular matrix, fibronectin and collagen-I was examined by quantitative real-time PCR and ELISA. F13A, an antagonist of the apelin receptor APJ, and small interfering RNA targeting protein kinase C epsilon (PKC-ε) were used to explore the relevant signaling pathways. Apelin attenuated TGF-β1-induced EMT, and inhibited the EMT-associated increase in α-SMA, loss of E-cadherin, and secretion of extracellular matrix. Moreover, apelin activated PKC-ε in tubular epithelial cells, which in turn decreased phospho-Smad2/3 levels and increased Smad-7 levels. APJ inhibition or PKC-ε deletion diminished apelin-induced modulation of Smad signaling and suppression of tubular EMT. Our findings identify a novel PKC-ε-dependent mechanism in which apelin suppresses TGF-β1-mediated activation of Smad signaling pathways and thereby inhibits tubular EMT. These results suggest that apelin may be a new agent that can suppress renal fibrosis and retard chronic kidney disease progression. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Aluminum phthalocyanine tetrasulfonate in MCF-10F, human breast epithelial cells: a hole burning study.

    PubMed Central

    Milanovich, N; Reinot, T; Hayes, J M; Small, G J

    1998-01-01

    Laser-induced holes are burned in the absorption spectrum of aluminum phthalocyanine tetrasulfonate (APT) in MCF-10F, human breast epithelial cells. The hole burning mechanism is shown to be nonphotochemical. The fluorescence excitation spectra and hole spectra are compared with those of APT in hyperquenched glassy films of water, ethanol, and methanol. The results show that the APT is in an acidic, aqueous environment with a hydrogen-bonded network similar to that of glassy water, but showing the influence of other cellular components. Pressure shifts of holes allow the local compressibility about the APT to be determined. PMID:9591692

  14. Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus.

    PubMed

    O'Hara, Bethany A; Gee, Gretchen V; Atwood, Walter J; Haley, Sheila A

    2018-04-15

    JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML. IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood

  15. Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

    NASA Technical Reports Server (NTRS)

    Tlsty, T. D.; Romanov, S. R.; Kozakiewicz, B. K.; Holst, C. R.; Haupt, L. M.; Crawford, Y. G.

    2001-01-01

    The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

  16. Bacterial Fimbriae and Their Peptides Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2

    PubMed Central

    Asai, Yasuyuki; Ohyama, Yoshinori; Gen, Keika; Ogawa, Tomohiko

    2001-01-01

    Gingival epithelial cells are a central component of the barrier between oral microflora and internal tissues. Host responses to periodontopathic bacteria and surface components containing fimbriae are thought to be important in the development and progression of periodontal diseases. To elucidate this mechanism, we established immortalized human gingival epithelial cells (HGEC) that were transfected with human papillomavirus. HGEC predominantly expressed Toll-like receptor (TLR) 2, but not TLR4 or CD14. They also induced interleukin-8 (IL-8) production when stimulated with Porphyromonas gingivalis fimbriae and Staphylococcus aureus peptidoglycan, but not Escherichia coli-type synthetic lipid A. Furthermore, an active synthetic peptide composed of residues 69 to 73 (ALTTE) of the fimbrial subunit protein, derived from P. gingivalis and similar to a common component of cell wall peptidoglycans in parasitic bacteria, N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), significantly induced IL-8 production and NF-κB activation in HGEC, and these cytokine-producing activities were augmented by a complex of soluble CD14 and lipopolysaccharide-binding protein (LBP). IL-8 production in HGEC stimulated with these bacterial components was clearly inhibited by mouse monoclonal antibody to human TLR2. These findings suggest that P. gingivalis fimbrial protein and its active peptide are capable of activating HGEC through TLR2. PMID:11705912

  17. Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential

    NASA Technical Reports Server (NTRS)

    Piao, C. Q.; Willey, J. C.; Hei, T. K.; Hall, E. J. (Principal Investigator)

    1999-01-01

    The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.

  18. Interleukin-33 induces mucin gene expression and goblet cell hyperplasia in human nasal epithelial cells.

    PubMed

    Ishinaga, Hajime; Kitano, Masako; Toda, Masaaki; D'Alessandro-Gabazza, Corina N; Gabazza, Esteban C; Shah, Said Ahmad; Takeuchi, Kazuhiko

    2017-02-01

    We investigated whether IL-33 is involved in mucus overproduction and goblet cell hyperplasia in eosinophilic chronic rhinosinusitis (ECRS). IL-33 mRNA was significantly higher in the eosinophilic CRS group than in the non-eosinophilic CRS group from human nasal polyps. IL-33 induced MUC5AC mRNA and MUC5AC protein, and also goblet cell hyperplasia at air liquid interface culture in human nasal epithelial cells. In addition to that, IL-33 induced MUC5B and FOXA3, and reduces FOXJmRNA. In conclusion, our present study demonstrated that the direct evidence of IL-33 which lead to increase mucin gene and protein expression, as well as goblet cell hyperplasia. This study provides novel insights into the role of IL-33 on mucus overproduction in eosinophilic inflammation of human airways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Human Skin Is the Largest Epithelial Surface for Interaction with Microbes.

    PubMed

    Gallo, Richard L

    2017-06-01

    Human skin contains an abundant and diverse population of microbial organisms. Many of these microbes inhabit follicular structures of the skin. Furthermore, numerous studies have shown that the interaction of some members of the skin microbiome with host cells will result in changes in cell function. However, estimates of the potential for the microbiome to influence human health through skin have ignored the inner follicular surface, and therefore vastly underestimated the potential of the skin microbiome to have a systemic effect on the human body. By calculating the surface area of follicular and the interfollicular epithelial surface it is shown that skin provides a vast interface for interactions with the microbiome. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  20. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.

    PubMed

    Meller, D; Pires, R T F; Tseng, S C G

    2002-04-01

    Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous

  1. Pro-inflammatory NF-κB and early growth response gene 1 regulate epithelial barrier disruption by food additive carrageenan in human intestinal epithelial cells.

    PubMed

    Choi, Hye Jin; Kim, Juil; Park, Seong-Hwan; Do, Kee Hun; Yang, Hyun; Moon, Yuseok

    2012-06-20

    The widely used food additive carrageenan (CGN) has been shown to induce intestinal inflammation, ulcerative colitis-like symptoms, or neoplasm in the gut epithelia in animal models, which are also clinical features of human inflammatory bowel disease. In this study, the effects of CGN on pro-inflammatory transcription factors NF-κB and early growth response gene 1 product (EGR-1) were evaluated in terms of human intestinal epithelial barrier integrity. Both pro-inflammatory transcription factors were elevated by CGN and only NF-κB activation was shown to be involved in the induction of pro-inflammatory cytokine interleukin-8. Moreover, the integrity of the in vitro epithelial monolayer under the CGN insult was maintained by both activated pro-inflammatory transcription factors NF-κB and EGR-1. Suppression of NF-κB or EGR-1 aggravated barrier disruption by CGN, which was associated with the reduced gene expression of tight junction component zonula occludens 1 and its irregular localization in the epithelial monolayer. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells.

    PubMed

    Pistone Creydt, Virginia; Fletcher, Sabrina Johanna; Giudice, Jimena; Bruzzone, Ariana; Chasseing, Norma Alejandra; Gonzalez, Eduardo Gustavo; Sacca, Paula Alejandra; Calvo, Juan Carlos

    2013-02-01

    Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines. Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post hoc tests were performed. Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs. We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.

  3. Focal epithelial hyperplasia in a human immuno-deficiency virus patient treated with laser surgery.

    PubMed

    Galanakis, Alexandros; Palaia, Gaspare; Tenore, Gianluca; Vecchio, Alessandro Del; Romeo, Umberto

    2014-07-16

    Focal epithelial hyperplasia (FEH), or Heck's disease, is a rare disease of the oral mucosa; it is mostly found in children or young adults who are immunosuppressed and who live in regions with low socioeconomic status. It is characterized by asymptomatic papules on the oral mucosa, gingiva, tongue, and lips. Healing can be spontaneous, and treatment is indicated if there are aesthetic or functional complications. Human papillomavirus, especially genotypes 13 and 32, has been associated with FEH and is detected in the majority of lesions. Histopathologically, FEH is characterized by parakeratosis, epithelial hyperplasia, focal acanthosis, and fusion and horizontal outgrowth of epithelial ridges. A 37-year-old male patient was referred to the Department of Oral and Maxillofacial Sciences at the Sapienza University of Rome, complaining of numerous exophytic lesions in his mouth. He stated that the lesions were not painful but he had experienced occasional bleeding after incidental masticatory trauma. He had received no previous treatment for the oral lesions. His medical history revealed that he was human immuno-deficiency virus positive and was a smoker with numerous, asymptomatic oral papules clinically and histologically corresponding to FEH. The labial and buccal mucosa were especially affected by lesions. Surgical treatment was performed using a 532-nm potassium titanyl phosphate laser (SmartLite, Deka, Florence, Italy) in continuous mode with a 300 μm fiber and power of 1.4 W (power density 1980.22 W/cm(2)). After anesthesia without vasoconstrictors, the lesions were tractioned with sutures or an Allis clamp and then completely excised. The lesions were preserved in 10% formalin for histological examination, which confirmed the clinical diagnosis of FEH. In this case, the laser allowed excellent control of bleeding, without postoperative sutures, and optimal wound healing.

  4. Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy.

    PubMed

    Guthoff, Rudolf F; Wienss, Holger; Hahnel, Christian; Wree, Andreas

    2005-07-01

    Evaluation of a new method to visualize distribution and morphology of human corneal nerves (Adelta- and C-fibers) by means of fluorescence staining, confocal laser scanning microscopy, and 3-dimensional (3D) reconstruction. Trephinates of corneas with a diagnosis of Fuchs corneal dystrophy were sliced into layers of 200 microm thickness using a Draeger microkeratome (Storz, Germany). The anterior lamella was stained with the Life/Dead-Kit (Molecular Probes Inc.), examined by the confocal laser scanning microscope "Odyssey XL," step size between 0.5 and 1 microm, and optical sections were digitally 3D-reconstructed. Immediate staining of explanted corneas by the Life/Dead-Kit gave a complete picture of the nerves in the central human cornea. Thin nerves running parallel to the Bowman layer in the subepithelial plexus perforate the Bowman layer orthogonally through tube-like structures. Passing the Bowman layer, Adelta- and C-fibers can be clearly distinguished by fiber diameter, and, while running in the basal epithelial plexus, by their spatial arrangement. Adelta-fibers run straight and parallel to the Bowman layer underneath the basal cell layer. C-fibers, after a short run parallel to the Bowman layer, send off multiple branches penetrating epithelial cell layers orthogonally, ending blindly in invaginations of the superficial cells. In contrast to C-fibers, Adelta-fibers show characteristic bulbous formations when kinking into the basal epithelial plexus. Ex-vivo fluorescence staining of the cornea and 3D reconstructions of confocal scans provide a fast and easily reproducible tool to visualize nerves of the anterior living cornea at high resolution. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

  5. Cholesterol depletion in cell membranes of human airway epithelial cells suppresses MUC5AC gene expression.

    PubMed

    Song, Kee Jae; Kim, Na Hyun; Lee, Gi Bong; Kim, Ji Hoon; Kwon, Jin Ho; Kim, Kyung-Su

    2013-05-01

    If cholesterol in the cell membrane is depleted by treating cells with methyl-β-cyclodextrin (MβCD), the activities of transmembrane receptors are altered in a cell-specific and/or receptor-specific manner. The proinflammatory cytokines, IL-1β is potent inducers of MUC5AC mRNA and protein synthesis in human airway epithelial cells. Cells activated by IL-1β showed increased phosphorylation of extracellular signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Thus, we investigated the effects of cholesterol depletion on the expression of MUC5AC in human airway epithelial cells and whether these alterations to MUC5AC expression were related to MAPK activity. After NCI-H292 cells were pretreated with 1% MβCD before adding IL-1β for 24 hours, MUC5AC mRNA expression was determined by reverse transcription- polymerase chain reaction (RT-PCR) and real time-PCR. Cholesterol depletion by MβCD was measured by modified microenzymatic fluorescence assay and filipin staining. The phosphorylation of IL-1 receptor, ERK and p38 MAPK, was analyzed by western blot. Cholesterol in the cell membrane was significantly depleted by treatment with MβCD on cells. IL-1β-induced MUC5AC mRNA expression was decreased by MβCD and this decrease occurred IL-1-receptor- specifically. Moreover, we have shown that MβCD suppressed the activation of ERK1/2 and p38 MAPK in cells activated with IL-1β. This result suggests that MβCD-mediated suppression of IL-1β-induced MUC5AC mRNA operated via the ERK- and p38 MAPK-dependent pathway. Cholesterol depletion in NCI-H292 cell membrane may be considered an anti-hypersecretory method since it effectively inhibits mucus secretion of respiratory epithelial cells.

  6. Mitochondrial superoxide dismutase activation with 17 β-estradiol-treated human lens epithelial cells

    PubMed Central

    Gottipati, Srinivas

    2008-01-01

    Purpose 17 β-estradiol (17β-E2) protects human lens epithelial cells against oxidative stress by preserving mitochondrial function in part via the non-genomic rapid activation of prosurvival signal transduction pathways. The study described herein examined whether 17β-E2 also elicits genomic protection by influencing the expression (and activity) of mitochondrial-associated manganese superoxide dismutase (MnSOD) as a possible parallel mechanism by which 17β-E2 protects against oxidative stress. Methods Virally-transformed human lens epithelial cells (HLE-B3) were pre-incubated with 17β-E2, and mRNA or protein lysates were collected over a time course ranging from 90 min to 24 h. Positive expression of lens epithelial cell MnSOD mRNA was determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT–PCR), and its levels were monitored by real-time PCR up to 24 h after 17β-E2 administration. Western blot analysis was used to examine the pattern of protein expression as influenced by 17β-E2 treatment. MnSOD activity as influenced by 17β-E2 was determined by measuring enzymatic activity. Results A significant rapid increase in the activity of MnSOD was observed with HLE-B3 cells by 90 min post-bolus addition of 17β-E2, which returned to control level by 240 min. Neither an increase in MnSOD mRNA nor in protein expression was detected up through 24 h. Conclusions These data demonstrate that 17β-E2 rapidly and transiently increases the activity of MnSOD but influences neither its mRNA expression nor its protein expression. The results suggest that (estrogen-activated) MnSOD plays an important role against mitochondrial oxidative stress by diminishing reactive oxygen species, thus promoting cell survival. PMID:18490963

  7. Telomerase activation by c-Myc in human mammary epithelial cells requires additional genomic changes.

    PubMed

    Bazarov, Alexey V; Hines, William C; Mukhopadhyay, Rituparna; Beliveau, Alain; Melodyev, Sonya; Zaslavsky, Yuri; Yaswen, Paul

    2009-10-15

    A central question in breast cancer biology is how cancer cells acquire telomerase activity required for unlimited proliferation. According to one model, proliferation of telomerase(-) pre-malignant cells leads to telomere dysfunction and increased genomic instability. Such instability leads in rare cases to reactivation of telomerase and immortalization. The mechanism of telomerase reactivation remains unknown. We have studied immortalization of cultured human mammary epithelial cells by c-Myc, a positive transcriptional regulator of the hTERT gene encoding the catalytic subunit of telomerase. Retrovirally introduced c-Myc cDNA resulted in immortalization of human mammary epithelial cells in which the cyclin dependent kinase inhibitor, p16(INK4A), was inactivated by an shRNA-encoding retrovirus. However, while c-Myc introduction immediately resulted in increased activity of transiently transfected hTERT promoter reporter constructs, endogenous hTERT mRNA levels did not change until about 60 population doublings after c-Myc introduction. Increased endogenous hTERT transcripts and stabilization of telomeric DNA in cells expressing exogenous c-Myc coincided with telomere dysfunction-associated senescence in control cultures. Genome copy number analyses of immortalized cells indicated amplifications of some or all of chromosome 5, where hTERT genes are located. hTERT gene copy number, however, was not increased in one case. The results are consistent with the hypothesis that changes in chromosome 5, while not necessarily increasing hTERT gene copy number, resulted in removal of repressive chromatin structures around hTERT loci, allowing induction of hTERT transcription. These in vitro results model one possible sequence of events leading to immortalization of breast epithelial cells during cancer progression.

  8. Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice

    PubMed Central

    Nilforoushzadeh, Mohammadali; Rahimi Jameh, Elham; Jaffary, Fariba; Abolhasani, Ehsan; Keshtmand, Gelavizh; Zarkob, Hajar; Mohammadi, Parvaneh; Aghdami, Nasser

    2017-01-01

    Objective Dermal papilla and hair epithelial stem cells regulate hair formation and the growth cycle. Damage to or loss of these cells can cause hair loss. Although several studies claim to reconstitute hairs using rodent cells in an animal model, additional research is needed to develop a stable human hair follicle reconstitution protocol. In this study, we have evaluated hair induction by injecting adult cultured human dermal papilla cells and a mixture of hair epithelial and dermal papilla cells in a mouse model. Materials and Methods In this experimental study, discarded human scalp skins were used to obtain dermal papilla and hair epithelial cells. After separation, cells were cultured and assessed for their characteristics. We randomly allocated 15 C57BL/6 nude mice into three groups that received injections in their dorsal skin. The first group received cultured dermal papilla cells, the second group received a mixture of cultured epithelial and dermal papilla cells, and the third group (control) received a placebo [phosphate-buffered saline (PBS-)]. Results Histopathologic examination of the injection sites showed evidence of hair growth in samples that received cells compared with the control group. However, the group that received epithelial and dermal papilla cells had visible evidence of hair growth. PKH tracing confirmed the presence of transplanted cells in the new hair. Conclusion Our data showed that injection of a combination of adult human cultured dermal papilla and epithelial cells could induce hair growth in nude mice. This study emphasized that the combination of human adult cultured dermal papilla and epithelial cells could induce new hair in nude mice. PMID:28670518

  9. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

    PubMed

    Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

    2015-09-01

    Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers. Copyright © 2015. Published by Elsevier B.V.

  10. [Expression of thioredoxin-2 in human lens epithelial cells with oxidative damage and its significance].

    PubMed

    Che, Xuanyi; Zhao, Qingxia; Li, Di

    2018-03-28

    To explore whether thioredoin-2 (Trx-2) is involved in the development of cataract and to study the effect of Trx-2 on hydrogen peroxide (H2O2)-induced injury in human lens epithelial cells.
 Methods: A total of 10 volunteers (removing the lens due totraumatism) and 30 patients received phacoemulsification (age more than 60 years) were selected. The expression of Trx-2 protein in lens epithelial cells from cataract patients and volunteers were detected by the immunohistochemical streptavidin-peroxidase (SP) method. SRA01/04 cells were cultured and were divided into six groups according to different treatment: a control group, H2O2-treated groups at 20, 50 or 
100 μmol/L, a negative control group (transfected with pCMV6 plasmid plus 100 μmol/L H2O2), and a Trx-2 overexpression group (transfected with pCMV6-Trx-2 plasmid plus 100 μmol/L H2O2). Methyl thiazolyltetrazolium (MTT) assay and flow cytometry was performed to measure the cell viability and apoptosis for SRA01/04 cells, respectively. The activities of superoxide dismutase (SOD) and catalase (CAT), the content of glutathione (GSH) and malondialdehyde (MDA) in human lens epithelial cells were measured via chemical chromatometry. Western blot was used to measure the protein levels of Trx-2, B-cell lymphoma 2 protein (Bcl-2), Bcl-2 associated X protein (Bax) and caspase-3.
 Results: Compared with the volunteers, the expression of Trx-2 was significantly decreased in lens epithelial cells in patients with cataract (P<0.05). Compared with the control group, the expression of Trx-2 protein in the 20, 50 or 100 μmol/L H2O2 groups was decreased (all P<0.05). Compared with the control group, the cell survival rates were decreased in the 100 μmol/L H2O2 group and the negative control group (both P<0.05), along with enhanced apoptotic rates, inhibited cellular SOD activities and CAT activities, reduced GSH contents, augmented MDA contents, down-regulated Trx-2 and Bcl-2 expression and up-regulated Bax and

  11. Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy

    NASA Astrophysics Data System (ADS)

    Adur, Javier; Pelegati, Vitor B.; de Thomaz, Andre A.; D'Souza-Li, Lilia; Assunção, Maria do Carmo; Bottcher-Luiz, Fátima; Andrade, Liliana A. L. A.; Cesar, Carlos L.

    2012-08-01

    We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.

  12. Chlorobenzene induces oxidative stress in human lung epithelial cells in vitro

    SciTech Connect

    Feltens, Ralph, E-mail: ralph.feltens@ufz.d; UFZ- Helmholtz Centre for Environmental Research, Department of Proteomics, Permoserstrasse 15, D-04318 Leipzig; Moegel, Iljana, E-mail: iljana.moegel@ufz.d

    Chlorobenzene is a volatile organic compound (VOC) that is widely used as a solvent, degreasing agent and chemical intermediate in many industrial settings. Occupational studies have shown that acute and chronic exposure to chlorobenzene can cause irritation of the mucosa of the upper respiratory tract and eyes. Using in vitro assays, we have shown in a previous study that human bronchial epithelial cells release inflammatory mediators such as the cytokine monocyte chemoattractant protein-1 (MCP-1) in response to chlorobenzene. This response is mediated through the NF-kappaB signaling pathway. Here, we investigated the effects of monochlorobenzene on human lung cells, with emphasismore » on potential alterations of the redox equilibrium to clarify whether the chlorobenzene-induced inflammatory response in lung epithelial cells is caused via an oxidative stress-dependent mechanism. We found that expression of cellular markers for oxidative stress, such as heme oxygenase 1 (HO-1), glutathione S-transferase pi1 (GSTP1), superoxide dismutase 1 (SOD1), prostaglandin-endoperoxide synthase 2 (PTGS2) and dual specificity phosphatase 1 (DUSP1), were elevated in the presence of monochlorobenzene. Likewise, intracellular reactive oxygen species (ROS) were increased in response to exposure. However, in the presence of the antioxidants N-(2-mercaptopropionyl)-glycine (MPG) or bucillamine, chlorobenzene-induced upregulation of marker proteins and release of the inflammatory mediator MCP-1 are suppressed. These results complement our previous findings and point to an oxidative stress-mediated inflammatory response following chlorobenzene exposure.« less

  13. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

    SciTech Connect

    Wu, Feng; Jordan, Ashley; Kluz, Thomas

    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 revealedmore » 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. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.« less

  14. Presence of melanopsin in human crystalline lens epithelial cells and its role in melatonin synthesis.

    PubMed

    Alkozi, Hanan Awad; Wang, Xiaoyu; Perez de Lara, Maria J; Pintor, Jesus

    2017-01-01

    Melanopsin is a non-image forming photoreceptor known to be present in the retina and it is considered to have light regulated tasks among other functions. In the present work, melanopsin presence in human lens epithelial cells as well as in human lens tissue is described for the first time. Moreover, studying the concentration of melatonin and its synthesising enzyme AANAT proved a clear link between melanopsin activation and the suppression of melatonin synthesis. Melanopsin sensitivity to specific wavelength (465-480 nm, blue) was confirmed after making temporal studies incubating lens epithelial cells under light, red, green, blue and total darkness for 2, 4, 8, 12 h and analysing the concentration of both melatonin and its synthesising enzyme AANAT, discovering that melatonin levels after submitting cells to total darkness are significantly higher to ones submitted to white or specifically blue light (***p < 0.001, n = 6). The involvement of melanopsin in the regulation of melatonin was also determined by using a specific inhibitor AA92593 and by inhibiting melanopsin-induced phospholipase C activation. Under this situation neither AANAT nor melatonin levels changed under light conditions (n = 4, ***p < 0.001). The discovery of melanopsin in the lens opens the possibility of regulating melatonin synthesis with the corresponding implication as an antioxidant substance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

    PubMed Central

    Zaheer, R S; Wiehler, S; Hudy, M H; Traves, S L; Pelikan, J B; Leigh, R; Proud, D

    2014-01-01

    Human rhinovirus (HRV) infections trigger exacerbations of lower airway diseases. HRV infects human airway epithelial cells and induces proinflammatory and antiviral molecules that regulate the response to HRV infection. Interferon (IFN)-stimulated gene of 15 kDa (ISG15) has been shown to regulate other viruses. We now show that HRV-16 infection induces both intracellular epithelial ISG15 expression and ISG15 secretion in vitro. Moreover, ISG15 protein levels increased in nasal secretions of subjects with symptomatic HRV infections. HRV-16-induced ISG15 expression is transcriptionally regulated via an IFN regulatory factor pathway. ISG15 does not directly alter HRV replication but does modulate immune signaling via the viral sensor protein RIG-I to impact production of CXCL10, which has been linked to innate immunity to viruses. Extracellular ISG15 also alters CXCL10 production. We conclude that ISG15 has a complex role in host defense against HRV infection, and that additional studies are needed to clarify the role of this molecule. PMID:24448099

  16. Vaccination with human amniotic epithelial cells confer effective protection in a murine model of Colon adenocarcinoma.

    PubMed

    Tabatabaei, M; Mosaffa, N; Ghods, R; Nikoo, S; Kazemnejad, S; Khanmohammadi, M; Mirzadeghan, E; Mahmoudi, A R; Bolouri, M R; Falak, R; Keshavarzi, B; Ramezani, M; Zarnani, A H

    2018-04-01

    As a prophylactic cancer vaccine, human amniotic membrane epithelial cells (hAECs) conferred effective protection in a murine model of colon cancer. The immunized mice mounted strong cross-protective CTL and antibody responses. Tumor burden was significantly reduced in tumor-bearing mice after immunization with hAECs. Placental cancer immunotherapy could be a promising approach for primary prevention of cancer. In spite of being the star of therapeutic strategies for cancer treatment, the results of immunotherapeutic approaches are still far from expectations. In this regard, primary prevention of cancer using prophylactic cancer vaccines has gained considerable attention. The immunologic similarities between cancer development and placentation have helped researchers to unravel molecular mechanisms responsible for carcinogenesis and to take advantage of stem cells from reproductive organs to elicit robust anti-cancer immune responses. Here, we showed that vaccination of mice with human amniotic membrane epithelial cells (hAECs) conferred effective protection against colon cancer and led to expansion of systemic and splenic cytotoxic T cell population and induction of cross-protective cytotoxic responses against tumor cells. Vaccinated mice mounted tumor-specific Th1 responses and produced cross-reactive antibodies against cell surface markers of cancer cells. Tumor burden was also significantly reduced in tumor-bearing mice immunized with hAECs. Our findings pave the way for potential future application of hAECs as an effective prophylactic cancer vaccine. © 2017 UICC.

  17. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

    NASA Technical Reports Server (NTRS)

    Romanov, S. R.; Kozakiewicz, B. K.; Holst, C. R.; Stampfer, M. R.; Haupt, L. M.; Tlsty, T. D.

    2001-01-01

    Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

  18. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

  19. Frequency of micronucleus in oral epithelial cells after exposure to mate-tea in healthy humans

    PubMed Central

    Campagnoli, Eduardo B.; Milan, José R.; Reinheimer, Angélica; Masson, Maicon; Capella, Diogo L.

    2014-01-01

    Objectives: The aim of this study was to evaluate the possibility of technique simplification for cytology slides in order to evaluate the frequency of micronuclei (FMic) and conduct a experiment looking to know the FMic of oral epithelial cells of healthy volunteers exposed to mate tea (Ilex paraguarariensis). Material and Methods: This is a laboratorial and nonrandomized trial (quasi-experiment), where the nonusers subjects were exposed to mate-tea, consumed in the traditional way, two drinks, two times a day for a single week. Two cytology of exfoliated epithelial cells were obtained before and after the mate tea exposition. Results: The sample was composed by 10 volunteers. The age ranged from 18 to 33 years (Mean 23; SD5.5). The use of mate tea did not showed significant variation in the FMic (Wilcoxon Signed Ranks Test p= .24). Conclusions: The proposed technique simplification showed to be reliable, without losses when compared to the conventional technique and with the advantage of eliminate toxic substances, becoming simple and practical tool for research in dentistry. The acute exposure to mate tea did not induce an increase of FMic in exfoliated buccal cells of healthy nondrinkers and nonsmokers subjects and may not have genotoxic effect. More human studies are needed before a conclusion can be made on the oral carcinogenic risk of mate tea to humans. Key words:Micronucleus, Oral Cancer, Cytology, Mate tea, Ilex paraguariensis. PMID:24608213

  20. ZN2+-INDUCED IL-8 EXPRESSION INVOLVES AP-1, JNK, AND ERK ACTIVITIES IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Exposure to zinc-laden particulate matter (PM) in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zin...

  1. Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation

    USDA-ARS?s Scientific Manuscript database

    Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our objective was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretor...

  2. MSH3-deficiency initiates EMAST without oncogenic transformation of human colon epithelial cells.

    PubMed

    Campregher, Christoph; Schmid, Gerald; Ferk, Franziska; Knasmüller, Siegfried; Khare, Vineeta; Kortüm, Benedikt; Dammann, Kyle; Lang, Michaela; Scharl, Theresa; Spittler, Andreas; Roig, Andres I; Shay, Jerry W; Gerner, Christopher; Gasche, Christoph

    2012-01-01

    Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay. Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10(-4)) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.

  3. MSH3-Deficiency Initiates EMAST without Oncogenic Transformation of Human Colon Epithelial Cells

    PubMed Central

    Campregher, Christoph; Schmid, Gerald; Ferk, Franziska; Knasmüller, Siegfried; Khare, Vineeta; Kortüm, Benedikt; Dammann, Kyle; Lang, Michaela; Scharl, Theresa; Spittler, Andreas; Roig, Andres I.; Shay, Jerry W.; Gerner, Christopher; Gasche, Christoph

    2012-01-01

    Background/Aim Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. Methods HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay. Results Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10−4) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. Conclusions MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis. PMID

  4. Diosmin reduces cell viability of A431 skin cancer cells through apoptotic induction.

    PubMed

    Buddhan, Rajamanickam; Manoharan, Shanmugam

    2017-01-01

    Aim of the present study was to evaluate the in vitro cytotoxic potential of the diosmin in A431 skin cancer cells. The cytotoxic (anti-cell proliferative) potential of diosmin in A431 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell viability), dual staining (apoptotic induction), dichloro-dihydro-fluorescein diacetate assay (reactive oxygen species [ROS] generation), DNA fragmentation study, Western blotting analysis (apoptotic markers expression) and flow cytometry (cell cycle arrest). Diosmin reduced the cell viability of A431 cells in a dose-dependent fashion and the inhibitory concentration 50% value was attained at 45 μg/ml using MTT assay. Diosmin at a concentration of 45 μg/ml generated excessive ROS in A431 cells, as compared to untreated cells. Diosmin treated A431 cells also revealed multiple DNA fragments than the untreated cells. Diosmin upregulated the expression of p53, caspases 3 and 9 and downregulated the expression of Bcl-2, matrix metalloproteinases-2 and 9 in A431 cells. The cytotoxic or anti-cell proliferative potential of diosmin is due to its ROS-mediated apoptotic induction potential, as well as due to its role in the inhibition of invasion in the A431 cells.

  5. Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function.

    PubMed

    Nilsson, M; Husmark, J; Nilsson, B; Tisell, L E; Ericson, L E

    1996-10-01

    Epithelial properties of thyrocytes are difficult to maintain in conventional cell culture systems. We used bicameral chambers (Transwell) in attempts to establish a functional epithelium of thyrocytes of human origin. Thyroid follicle segments were isolated by collagenase digestion of paradenomatous tissue obtained at surgery for follicular adenoma and of tissue from glands with Graves' disease. After careful separation from connective tissue and single cells by centrifugation, the follicles were plated at high density on the collagen-coated filter of the chambers and cultured in Eagle's essential medium (EMEM) containing 10% fetal calf serum (FCS) or Coon's modified Hams medium enriched with five or six factors (5H, 6H); the latter media contained 5% FCS without (5H) or with (6H) thyrotropin (TSH). The follicles were converted into a confluent cell layer, which had similar DNA content irrespective of type of medium, after 4-6 days. Cells grown in EMEM or 5H established a transepithelial electrical resistance (R) of 200-500 omega.cm2 and was impermeable to [3H]inulin, indicating the formation of epithelial junctions. Addition of 6H to confluent cells initially cultured in EMEM or 5H caused a further increase of R, maximally to 1500 omega.cm2, along with a rise of the transepithelial potential difference; 6H promoted the monolayer formation of cells, increased the number of apical microvilli and reinforced the junctional distribution of actin, cadherin and ZO-1; 6H also enhanced the polarized secretion of [3H]leucine-labeled thyroglobulin into the apical medium. Cells from Graves' thyroid tissue established an epithelium on the filter with similar characteristics to that of normal thyrocytes; some platings contained in addition large numbers of HLA-DR positive cells with a dendritic shape. HLA-DR expression was generally absent in EMEM-or 5H-grown thyrocytes, but appeared in limited areas of the cell layer after 6H and was expressed by all epithelial cells after

  6. miR-300 inhibits epithelial to mesenchymal transition and metastasis by targeting Twist in human epithelial cancer

    PubMed Central

    2014-01-01

    Background Epithelial-to-mesenchymal transition (EMT) is a key step of the progression of tumor cell metastasis. Recent work has demonstrated some miRNAs play critical roles in EMT. In this study, we focused on the roles of miR-300 in regulating EMT. Methods The expression levels of miR-300 were examined in epithelial carcinoma cells that underwent an EMT using quantitative reverse transcription-PCR. The role of miR-300 in EMT was investigated by transfection of the miR-300 mimic or inhibitor in natural epithelial-mesenchymal phenotype cell line pairs and in transforming growth factor (TGF) beta-induced EMT cell models. A luciferase reporter assay and a rescue experiment were conducted to confirm the target gene of miR-300. The efficacy of miR-300 against tumor invasion and metastasis was evaluated both in vitro and in vivo. Correlation analysis between miR-300 expression and the expression levels of its target gene, as well as tumor metastasis was performed in specimens from patients with head and neck squamous cell carcinoma (HNSCC). Results MiR-300 was found down-regulated in the HNSCC cells and breast cancer cells that underwent EMT. Ectopic expression of miR-300 effectively blocked TGF-beta-induced EMT and reversed the phenotype of EMT in HN-12 and MDA-MB-231 cells, but inhibition of miR-300 in the epithelial phenotype cells, HN-4 and MCF-7 cells, could induce EMT. The luciferase reporter assay and the rescue assay results showed that miR-300 directly targets the 3′UTR of Twist. Enforced miR-300 expression suppressed cell invasion in vitro and experimental metastasis in vivo. Clinically, miR-300 expression was found inversely correlated with Twist expression and reduced miR-300 was associated with metastasis in patient specimens. Conclusions Down-regulation of miR-300 is required for EMT initiation and maintenance. MiR-300 may negatively regulate EMT by direct targeting Twist and therefore inhibit cancer cell invasion and metastasis, which implicates miR-300

  7. miR-300 inhibits epithelial to mesenchymal transition and metastasis by targeting Twist in human epithelial cancer.

    PubMed

    Yu, Jingshuang; Xie, Furong; Bao, Xin; Chen, Wantao; Xu, Qin

    2014-05-24

    Epithelial-to-mesenchymal transition (EMT) is a key step of the progression of tumor cell metastasis. Recent work has demonstrated some miRNAs play critical roles in EMT. In this study, we focused on the roles of miR-300 in regulating EMT. The expression levels of miR-300 were examined in epithelial carcinoma cells that underwent an EMT using quantitative reverse transcription-PCR. The role of miR-300 in EMT was investigated by transfection of the miR-300 mimic or inhibitor in natural epithelial-mesenchymal phenotype cell line pairs and in transforming growth factor (TGF) beta-induced EMT cell models. A luciferase reporter assay and a rescue experiment were conducted to confirm the target gene of miR-300. The efficacy of miR-300 against tumor invasion and metastasis was evaluated both in vitro and in vivo. Correlation analysis between miR-300 expression and the expression levels of its target gene, as well as tumor metastasis was performed in specimens from patients with head and neck squamous cell carcinoma (HNSCC). MiR-300 was found down-regulated in the HNSCC cells and breast cancer cells that underwent EMT. Ectopic expression of miR-300 effectively blocked TGF-beta-induced EMT and reversed the phenotype of EMT in HN-12 and MDA-MB-231 cells, but inhibition of miR-300 in the epithelial phenotype cells, HN-4 and MCF-7 cells, could induce EMT. The luciferase reporter assay and the rescue assay results showed that miR-300 directly targets the 3'UTR of Twist. Enforced miR-300 expression suppressed cell invasion in vitro and experimental metastasis in vivo. Clinically, miR-300 expression was found inversely correlated with Twist expression and reduced miR-300 was associated with metastasis in patient specimens. Down-regulation of miR-300 is required for EMT initiation and maintenance. MiR-300 may negatively regulate EMT by direct targeting Twist and therefore inhibit cancer cell invasion and metastasis, which implicates miR-300 as an attractive candidate for cancer

  8. Mechanisms and regulation of polymorphonuclear leukocyte and eosinophil adherence to human airway epithelial cells.

    PubMed

    Jagels, M A; Daffern, P J; Zuraw, B L; Hugli, T E

    1999-09-01

    Polymorphonuclear leukocytes (PMN) and eosinophils (Eos) are important cellular participants in a variety of acute and chronic inflammatory reactions in the airway. Histologic evidence has implicated direct interactions between these two subsets of leukocytes and airway epithelial cells during inflammation. A comprehensive characterization and comparison of physiologic stimuli and adhesion molecule involvement in granulocyte-epithelial-cell interactions done with nontransformed human airway epithelial cells has not been reported. We therefore examined the regulation and biochemical mechanisms governing granulocyte-epithelial-cell adhesion, using either purified PMN or Eos and primary cultures of human bronchial epithelial cells (HBECs). We investigated the involvement of a number of proinflammatory signals associated with allergic and nonallergic airway inflammation, as well as the contribution of several epithelial and leukocyte adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and members of the beta(1), beta(2), and beta(7) integrin families. ICAM-1 was expressed at low levels on cultured HBECs and was markedly upregulated after stimulation with interferon (IFN)-gamma or, to a lesser extent, with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1. VCAM-1 was not present on resting HBECs, and was not upregulated after stimulation with IFN-gamma, IL-1, IL-4, or TNF-alpha. PMN adhesion to HBECs could be induced either through activation of PMN with IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), or C5a, but not with IL-5 or by preactivation of HBECs with TNF-alpha or IFN-gamma. Blocking antibody studies indicated that PMN-HBEC adherence depended on beta(2) integrins, primarily alpha(M)beta(2) (Mac-1). Adherence of Eos to HBECs could be induced through activation of Eos with IL-5, GM-CSF, or C5a, but not with IL-8 or by prior activation of HBECs with TNF-alpha of IFN

  9. An Organotypic 3D Assay for Primary Human Mammary Epithelial Cells that Recapitulates Branching Morphogenesis.

    PubMed

    Linnemann, Jelena R; Meixner, Lisa K; Miura, Haruko; Scheel, Christina H

    2017-01-01

    We have developed a three-dimensional organotypic culture system for primary human mammary epithelial cells (HMECs) in which the cells are cultured in free floating collagen type I gels. In this assay, luminal cells predominantly form multicellular spheres, while basal/myoepithelial cells form complex branched structures resembling terminal ductal lobular units (TDLUs), the functional units of the human mammary gland in situ. The TDLU-like organoids can be cultured for at least 3 weeks and can then be passaged multiple times. Subsequently, collagen gels can be stained with carmine or by immunofluorescence to allow for the analysis of morphology, protein expression and polarization, and to facilitate quantification of structures. In addition, structures can be isolated for gene expression analysis. In summary, this technique is suitable for studying branching morphogenesis, regeneration, and differentiation of HMECs as well as their dependence on the physical environment.

  10. Cytotoxicity of protein corona-graphene oxide nanoribbons on human epithelial cells

    NASA Astrophysics Data System (ADS)

    Mbeh, Doris A.; Akhavan, Omid; Javanbakht, Taraneh; Mahmoudi, Morteza; Yahia, L.'Hocine

    2014-11-01

    Graphene oxide nanoribbons (GONRs) were synthesized using an oxidative unzipping of multi-walled carbon nanotubes. The interactions of the GONRs with various concentrations of fetal bovine serum or human plasma serum indicated that the GONRs were functionalized substantially by the albumin originated from the two different protein sources. Then, concentration-dependent cytotoxicity of the protein-functionalized GONRs on human epithelial cells was studied. Although the GONRs with concentrations ≤50 μg/mL did not exhibit significant cytotoxicity on the cells (with the cell viability >85%), the concentration of 100 μg/mL exhibited significant cytotoxicity including prevention of cell proliferation and induction of cell apoptosis. These results can provide more in-depth understanding about cytotoxic effects of graphene nanostructures which can be functionalized by the proteins of media.

  11. Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

    SciTech Connect

    Moonen, Harald J.J.; Geraets, Liesbeth; Vaarhorst, Anika

    2005-12-30

    Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzymemore » inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.« less

  12. Toward unraveling the complexity of simple epithelial keratins in human disease.

    PubMed

    Omary, M Bishr; Ku, Nam-On; Strnad, Pavel; Hanada, Shinichiro

    2009-07-01

    Simple epithelial keratins (SEKs) are found primarily in single-layered simple epithelia and include keratin 7 (K7), K8, K18-K20, and K23. Genetically engineered mice that lack SEKs or overexpress mutant SEKs have helped illuminate several keratin functions and served as important disease models. Insight into the contribution of SEKs to human disease has indicated that K8 and K18 are the major constituents of Mallory-Denk bodies, hepatic inclusions associated with several liver diseases, and are essential for inclusion formation. Furthermore, mutations in the genes encoding K8, K18, and K19 predispose individuals to a variety of liver diseases. Hence, as we discuss here, the SEK cytoskeleton is involved in the orchestration of several important cellular functions and contributes to the pathogenesis of human liver disease.

  13. Toward unraveling the complexity of simple epithelial keratins in human disease

    PubMed Central

    Omary, M. Bishr; Ku, Nam-On; Strnad, Pavel; Hanada, Shinichiro

    2009-01-01

    Simple epithelial keratins (SEKs) are found primarily in single-layered simple epithelia and include keratin 7 (K7), K8, K18–K20, and K23. Genetically engineered mice that lack SEKs or overexpress mutant SEKs have helped illuminate several keratin functions and served as important disease models. Insight into the contribution of SEKs to human disease has indicated that K8 and K18 are the major constituents of Mallory-Denk bodies, hepatic inclusions associated with several liver diseases, and are essential for inclusion formation. Furthermore, mutations in the genes encoding K8, K18, and K19 predispose individuals to a variety of liver diseases. Hence, as we discuss here, the SEK cytoskeleton is involved in the orchestration of several important cellular functions and contributes to the pathogenesis of human liver disease. PMID:19587454

  14. Role of human epididymis protein 4 in chemoresistance and prognosis of epithelial ovarian cancer.

    PubMed

    Lee, Seungho; Choi, Seowon; Lee, Yookyung; Chung, Donghae; Hong, Suntaek; Park, Nohhyun

    2017-01-01

    Human epididymis protein 4 (HE4) is a novel biomarker for epithelial ovarian cancer. This study was designed to evaluate the role of HE4 in chemo-response against anti-cancer drugs and prognosis of epithelial ovarian cancer. HE4-depleted cells and HE4-overexpressing cells were generated. The effect of HE4 gene silencing and overexpression was examined using a cell viability assay after exposure to chemotherapeutic agents and the signaling pathway. We studied the expression of HE4 in ovarian cancer tissue and the prognostic significance. Cytoplasmic staining was graded for intensity and percentage of positive cells. The grades were multiplied to determine an H-score. Knockdown of HE4 in OVCAR-3 cells resulted in reduction in cell growth and increased sensitivity to paclitaxel and cisplatin compared to control cells. This effect originated from the decreased activation of cell-growth-related signaling, such as AKT and Erk mediated by epidermal growth factor (EGF), while overexpression of HE4 resulted in enhanced cell growth and suppressed the anti-tumorigenic activity of paclitaxel. Activation of AKT and Erk pathways was enhanced in HE4-overexpressing cells compared to control cells. Based on the results of multivariate analysis, the risk of death was significantly higher in patients with an H-score > 4. HE4 induces chemoresistance against anti-cancer drugs and activates the AKT and Erk pathways to enhance tumor survival. HE4 expression in ovarian cancer tissue is associated with a worse prognosis for epithelial ovarian cancer patients. © 2016 Japan Society of Obstetrics and Gynecology.

  15. Cigarette smoke differentially affects IL-13-induced gene expression in human airway epithelial cells.

    PubMed

    Mertens, Tinne C J; van der Does, Anne M; Kistemaker, Loes E; Ninaber, Dennis K; Taube, Christian; Hiemstra, Pieter S

    2017-07-01

    Allergic airways inflammation in asthma is characterized by an airway epithelial gene signature composed of POSTN , CLCA1 , and SERPINB2 This Th2 gene signature is proposed as a tool to classify patients with asthma into Th2-high and Th2-low phenotypes. However, many asthmatics smoke and the effects of cigarette smoke exposure on the epithelial Th2 gene signature are largely unknown. Therefore, we investigated the combined effect of IL-13 and whole cigarette smoke (CS) on the Th2 gene signature and the mucin-related genes MUC5AC and SPDEF in air-liquid interface differentiated human bronchial (ALI-PBEC) and tracheal epithelial cells (ALI-PTEC). Cultures were exposed to IL-13 for 14 days followed by 5 days of IL-13 with CS exposure. Alternatively, cultures were exposed once daily to CS for 14 days, followed by 5 days CS with IL-13. POSTN , SERPINB2 , and CLCA1 expression were measured 24 h after the last exposure to CS and IL-13. In both models POSTN , SERPINB2 , and CLCA1 expression were increased by IL-13. CS markedly affected the IL-13-induced Th2 gene signature as indicated by a reduced POSTN , CLCA1 , and MUC5AC expression in both models. In contrast, IL-13-induced SERPINB2 expression remained unaffected by CS, whereas SPDEF expression was additively increased. Importantly, cessation of CS exposure failed to restore IL-13-induced POSTN and CLCA1 expression. We show for the first time that CS differentially affects the IL-13-induced gene signature for Th2-high asthma. These findings provide novel insights into the interaction between Th2 inflammation and cigarette smoke that is important for asthma pathogenesis and biomarker-guided therapy in asthma. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  16. Acute Lung Injury Edema Fluid Decreases Net Fluid Transport across Human Alveolar Epithelial Type II Cells*

    PubMed Central

    Lee, Jae W.; Fang, Xiaohui; Dolganov, Gregory; Fremont, Richard D.; Bastarache, Julie A.; Ware, Lorraine B.; Matthay, Michael A.

    2009-01-01

    Most patients with acute lung injury (ALI) have reduced alveolar fluid clearance that has been associated with higher mortality. Several mechanisms may contribute to the decrease in alveolar fluid clearance. In this study, we tested the hypothesis that pulmonary edema fluid from patients with ALI might reduce the expression of ion transport genes responsible for vectorial fluid transport in primary cultures of human alveolar epithelial type II cells. Following exposure to ALI pulmonary edema fluid, the gene copy number for the major sodium and chloride transport genes decreased. By Western blot analyses, protein levels of αENaC, α1Na,K-ATPase, and cystic fibrosis transmembrane conductance regulator decreased as well. In contrast, the gene copy number for several inflammatory cytokines increased markedly. Functional studies demonstrated that net vectorial fluid transport was reduced for human alveolar type II cells exposed to ALI pulmonary edema fluid compared with plasma (0.02±0.05 versus 1.31±0.56 μl/cm2/h, p<0.02). An inhibitor of p38 MAPK phosphorylation (SB202190) partially reversed the effects of the edema fluid on net fluid transport as well as gene and protein expression of the main ion transporters. In summary, alveolar edema fluid from patients with ALI induced a significant reduction in sodium and chloride transport genes and proteins in human alveolar epithelial type II cells, effects that were associated with a decrease in net vectorial fluid transport across human alveolar type II cell monolayers. PMID:17580309

  17. Human airway epithelial cell cultures for modeling respiratory syncytial virus infection.

    PubMed

    Pickles, Raymond J

    2013-01-01

    Respiratory syncytial virus (RSV) is an important human respiratory pathogen with narrow species tropism. Limited availability of human pathologic specimens during early RSV-induced lung disease and ethical restrictions for RSV challenge studies in the lower airways of human volunteers has slowed our understanding of how RSV causes airway disease and greatly limited the development of therapeutic strategies for reducing RSV disease burden. Our current knowledge of RSV infection and pathology is largely based on in vitro studies using nonpolarized epithelial cell-lines grown on plastic or in vivo studies using animal models semipermissive for RSV infection. Although these models have revealed important aspects of RSV infection, replication, and associated inflammatory responses, these models do not broadly recapitulate the early interactions and potential consequences of RSV infection of the human columnar airway epithelium in vivo. In this chapter, the pro et contra of in vitro models of human columnar airway epithelium and their usefulness in respiratory virus pathogenesis and vaccine development studies will be discussed. The use of such culture models to predict characteristics of RSV infection and the correlation of these findings to the human in vivo situation will likely accelerate our understanding of RSV pathogenesis potentially identifying novel strategies for limiting the severity of RSV-associated airway disease.

  18. Pseudomonas Pyocyanin Increases Interleukin-8 Expression by Human Airway Epithelial Cells

    PubMed Central

    Denning, Gerene M.; Wollenweber, Laura A.; Railsback, Michelle A.; Cox, Charles D.; Stoll, Lynn L.; Britigan, Bradley E.

    1998-01-01

    Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1α. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease. PMID:9826354

  19. Human Corneal Limbal-Epithelial Cell Response to Varying Silk Film Geometric Topography In Vitro

    PubMed Central

    Lawrence, Brian D.; Pan, Zhi; Liu, Aihong; Kaplan, David L.; Rosenblatt, Mark I.

    2012-01-01

    Silk fibroin films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding silk films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal-epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that silk film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment, and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36%–54% increase on average in initial cell attachment, which corresponded to an over 2-fold increase in FA localization when compared to other silk film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned silk film topographies. In conclusion, silk film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially in regards to FA distribution, in vitro. PMID:22705042

  20. Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research

    PubMed Central

    Stewart, Ceri E.; Torr, Elizabeth E.; Mohd Jamili, Nur H.; Bosquillon, Cynthia; Sayers, Ian

    2012-01-01

    The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions. PMID:22287976

  1. Dienogest inhibits C-C motif chemokine ligand 20 expression in human endometriotic epithelial cells.

    PubMed

    Mita, Shizuka; Nakakuki, Masanori; Ichioka, Masayuki; Shimizu, Yutaka; Hashiba, Masamichi; Miyazaki, Hiroyasu; Kyo, Satoru

    2017-07-01

    C-C motif chemokine ligand 20 is thought to contribute to the development of endometriosis by recruiting Th17 lymphocytes into endometriotic foci. The present study investigated the effects of dienogest, a progesterone receptor agonist used to treat endometriosis, on C-C motif chemokine ligand 20 expression by endometriotic cells. Effects of dienogest on mRNA expression and protein secretion of C-C motif chemokine ligand 20 induced by interleukin 1β were assessed in three immortalized endometriotic epithelial cell lines, parental cells (EMosis-CC/TERT1), and stably expressing human progesterone receptor isoform A (EMosis-CC/TERT1/PRA+) or isoform B (EMosis-CC/TERT1/PRA-/PRB+). Dienogest markedly inhibited interleukin 1β-stimulated C-C motif chemokine ligand 20 mRNA expression and protein secretion in EMosis-CC/TERT1/PRA-/PRB+, which was abrogated by the progesterone receptor antagonist RU486. In EMosis-CC/TERT1/PRA+, dienogest slightly inhibited C-C motif chemokine ligand 20 mRNA and protein. In EMosis-CC/TERT1, dienogest slightly inhibited C-C motif chemokine ligand 20 mRNA, but had no effect on C-C motif chemokine ligand 20 protein. Dienogest inhibited interleukin 1β-induced up-regulation of C-C motif chemokine ligand 20 in endometriotic epithelial cells, mainly mediated by progesterone receptor B. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Epithelial-stromal interface in normal and neoplastic human bladder epithelium.

    PubMed

    Alroy, J; Gould, V E

    1980-01-01

    The ultrastructure of the epithelial-stromal interface of the human urinary bladder was studied in biopsy specimens that included 7 normal controls, 1 inverted papilloma, 18 noninvasive papillary carcinomas, and 19 invasive transitional cell carcinomas. In the invasive foci of the transitional cell carcinomas, the underlying basal lamina was attenuated or absent and the number of hemidesmosomes was decreased. These neoplastic cells displayed notably increased numbers of lysosomes, some of which appeared to be in the process of exocytosis. Increased numbers of cytoplasmic filaments adjacent to the plasma membranes at the invading pole of these cells were also observed. Tight junctions and junctional complexes were noticed adjacent to the tumor-stromal interface. None of the aforementioned features was observed in normal transitional epithelium, in inverted papilloma, in noninvasive papillary carcinomas, or in the noninvasive portions of invasive transitional cell carcinomas. Alterations of the epithelial-stromal interface deserve additional studies for they may constitute important parameters in the evaluation of actual or potential invasiveness in the various types of carcinoma of the bladder.

  3. Human mammary epithelial cells exhibit a bimodal correlated random walk pattern.

    PubMed

    Potdar, Alka A; Jeon, Junhwan; Weaver, Alissa M; Quaranta, Vito; Cummings, Peter T

    2010-03-10

    Organisms, at scales ranging from unicellular to mammals, have been known to exhibit foraging behavior described by random walks whose segments confirm to Lévy or exponential distributions. For the first time, we present evidence that single cells (mammary epithelial cells) that exist in multi-cellular organisms (humans) follow a bimodal correlated random walk (BCRW). Cellular tracks of MCF-10A pBabe, neuN and neuT random migration on 2-D plastic substrates, analyzed using bimodal analysis, were found to reveal the BCRW pattern. We find two types of exponentially distributed correlated flights (corresponding to what we refer to as the directional and re-orientation phases) each having its own correlation between move step-lengths within flights. The exponential distribution of flight lengths was confirmed using different analysis methods (logarithmic binning with normalization, survival frequency plots and maximum likelihood estimation). Because of the presence of non-uniform turn angle distribution of move step-lengths within a flight and two different types of flights, we propose that the epithelial random walk is a BCRW comprising of two alternating modes with varying degree of correlations, rather than a simple persistent random walk. A BCRW model rather than a simple persistent random walk correctly matches the super-diffusivity in the cell migration paths as indicated by simulations based on the BCRW model.

  4. Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

    PubMed

    Min, Kyoung Ah; Rosania, Gus R; Kim, Chong-Kook; Shin, Meong Cheol

    2016-03-01

    To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

  5. KCC2a expression in a human fetal lens epithelial cell line.

    PubMed

    Lauf, Peter K; Di Fulvio, Mauricio; Srivastava, Vinita; Sharma, Neelima; Adragna, Norma C

    2012-01-01

    The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin. Copyright © 2012 S. Karger AG, Basel.

  6. Ferulic Acid Suppresses Amyloid β Production in the Human Lens Epithelial Cell Stimulated with Hydrogen Peroxide

    PubMed Central

    Nagai, Noriaki; Kotani, Sachiyo; Mano, Yu; Ueno, Akina; Ito, Yoshimasa; Kitaba, Toshio; Takata, Takumi

    2017-01-01

    It is well known that oxidative stresses induce the production of amyloid β (Aβ) in the brain, lens, and retina, leading to age-related diseases. In the present study, we investigated the effects of ferulic acid on the Aβ levels in H2O2-stimulated human lens epithelial (HLE) SRA 01/04 cells. Three types of Aβ peptides (Aβ1-40, Aβ1-42, and Aβ1-43) were measured by ELISA, and the levels of mRNA for the expressed proteins related to Aβ production (APP, BACE1, and PS proteins) and degradation (ADAM10, NEP, and ECE1 proteins) were determined by quantitative real-time RT-PCR. H2O2 stimulation augmented gene expression of the proteins related to Aβ production, resulting in the production of three types of Aβ peptides. Treatment with 0.1 μM ferulic acid attenuated the augmentations of gene expression and production of the proteins related to the secretion of three types of Aβ peptides in the H2O2-stimulated HLE cells. These results provided evidence of antioxidative functions of ferulic acid for lens epithelial cells. PMID:28409157

  7. Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers

    PubMed Central

    Min, Kyoung Ah; Rosania, Gus R.; Kim, Chong-Kook; Shin, Meong Cheol

    2016-01-01

    To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies. PMID:26746641

  8. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

    SciTech Connect

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 {mu}M triggered cell differentiation towards a neuronal-like phenotype: cells emittedmore » filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.« less

  9. IL-27 Modulates Chemokine Production in TNF-α -Stimulated Human Oral Epithelial Cells.

    PubMed

    Hosokawa, Yoshitaka; Hosokawa, Ikuko; Ozaki, Kazumi; Matsuo, Takashi

    2017-01-01

    Interleukin-27 (IL-27) is a cytokine which belongs to the IL-12 family. However, the role of IL-27 in the pathogenesis of periodontal disease is uncertain. The aim of this study was to examine the effect of IL-27 on chemokine production in TNF-α-stimulated human oral epithelial cells (TR146). We measured chemokine production in TR146 by ELISA. We used western blot analysis to detect the phosphorylation levels of signal transduction molecules, including STAT1 and STAT3 in TR146. We used inhibitors to examine the role of STAT1 and STAT3 activation. IL-27 increased CXCR3 ligands production in TNF-α-stimulated TR146. Meanwhile, IL-27 suppressed IL-8 and CCL20 production induced by TNF-α. STAT1 phosphorylation level in IL-27 and TNF-α-stimulated TR146 was enhanced in comparison to TNF-α-stimulated TR146. STAT3 phosphorylation level in IL-27-treated TR146 did not change by TNF-α. Both STAT1 inhibitor and STAT3 inhibitor decreased CXCR3 ligands production. STAT1 inhibitor overrode the inhibitory effect of IL-27 on IL-8 and CCL20 production in TNF-α-stimulated TR146. Meanwhile, STAT3 inhibitor did not modulate IL-8 and CCL20 production. IL-27 might control leukocyte migration in periodontal lesion by modulating chemokine production from epithelial cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

  10. LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells.

    PubMed

    Reyes-Reyes, Elsa M; Aispuro, Ivan; Tavera-Garcia, Marco A; Field, Matthew; Moore, Sara; Ramos, Irma; Ramos, Kenneth S

    2017-11-28

    Although several lines of evidence have established the central role of epithelial-to-mesenchymal-transition (EMT) in malignant progression of non-small cell lung cancers (NSCLCs), the molecular events connecting EMT to malignancy remain poorly understood. This study presents evidence that Long Interspersed Nuclear Element-1 (LINE-1) retrotransposon couples EMT programming with malignancy in human bronchial epithelial cells (BEAS-2B). This conclusion is supported by studies showing that: 1) activation of EMT programming by TGF-β1 increases LINE-1 mRNAs and protein; 2) the lung carcinogen benzo(a)pyrene coregulates TGF-β1 and LINE-1 mRNAs, with LINE-1 positioned downstream of TGF-β1 signaling; and, 3) forced expression of LINE-1 in BEAS-2B cells recapitulates EMT programming and induces malignant phenotypes and tumorigenesis in vivo . These findings identify a TGFβ1-LINE-1 axis as a critical effector pathway that can be targeted for the development of precision therapies during malignant progression of intractable NSCLCs.

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

    PubMed Central

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

    2015-01-01

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

  12. Overexpression of Notch3 and pS6 Is Associated with Poor Prognosis in Human Ovarian Epithelial Cancer.

    PubMed

    Liu, Zhaoxia; Yun, Rongna; Yu, Xiaolin; Hu, Hui; Huang, Genhua; Tan, Buzhen; Chen, Tingtao

    2016-01-01

    Notch3 and pS6 play important roles in tumor angiogenesis. To assess the expression of Notch3 and pS6 in Chinese ovarian epithelial cancer patients, a ten-year follow-up study was performed in ovarian epithelial cancer tissues from 120 specimens of human ovarian epithelial cancer, 30 specimens from benign ovarian tumors, and 30 samples from healthy ovaries by immunohistochemistry. The results indicate that the expression of Notch3 and pS6 was higher in ovarian epithelial cancer than in normal ovary tissues and in benign ovarian tumor tissues (p < 0.01). In tumor tissues, Notch3 expression and pS6 expression were negatively associated with age (p > 0.05) but positively associated with clinical stage, pathological grading, histologic type, lymph node metastasis, and ascites (p < 0.05 or p < 0.01). A follow-up survey of 64 patients with ovarian epithelial cancer showed that patients with high Notch3 and pS6 expression had a shorter survival time (p < 0.01), in which the clinical stage (p < 0.05) and Notch3 expression (p < 0.01) played important roles. In conclusion, Notch3 and pS6 are significantly related to ovarian epithelial cancer development and prognosis, and their combination represents a potential biomarker and therapeutic target in ovarian tumor angiogenesis.

  13. Glucocorticoid action in human corneal epithelial cells establishes roles for corticosteroids in wound healing and barrier function of the eye.

    PubMed

    Kadmiel, Mahita; Janoshazi, Agnes; Xu, Xiaojiang; Cidlowski, John A

    2016-11-01

    Glucocorticoids play diverse roles in almost all physiological systems of the body, including both anti-inflammatory and immunosuppressive roles. Synthetic glucocorticoids are one of the most widely prescribed drugs and are used in the treatment of conditions such as autoimmune diseases, allergies, ocular disorders and certain types of cancers. In the interest of investigating glucocorticoid actions in the cornea of the eye, we established that multiple cell types in mouse corneas express functional glucocorticoid receptor (GR) with corneal epithelial cells having robust expression. To define glucocorticoid actions in a cell type-specific manner, we employed immortalized human corneal epithelial (HCE) cell line to define the glucocorticoid transcriptome and elucidated its functions in corneal epithelial cells. Over 4000 genes were significantly regulated within 6 h of dexamethasone treatment, and genes associated with cell movement, cytoskeletal remodeling and permeability were highly regulated. Real-time in vitro wound healing assays revealed that glucocorticoids delay wound healing by attenuating cell migration. These functional alterations were associated with cytoskeletal remodeling at the wounded edge of a scratch-wounded monolayer. However, glucocorticoid treatment improved the organization of tight-junction proteins and enhanced the epithelial barrier function. Our results demonstrate that glucocorticoids profoundly alter corneal epithelial gene expression and many of these changes likely impact both wound healing and epithelial cell barrier function. Published by Elsevier Ltd.

  14. Role of mesothelin in carbon nanotube-induced carcinogenic transformation of human bronchial epithelial cells

    PubMed Central

    He, Xiaoqing; Despeaux, Emily; Stueckle, Todd A.; Chi, Alexander; Castranova, Vincent; Dinu, Cerasela Zoica; Wang, Liying

    2016-01-01

    Carbon nanotubes (CNTs) have been likened to asbestos in terms of morphology and toxicity. CNT exposure can lead to pulmonary fibrosis and promotion of tumorigenesis. However, the mechanisms underlying CNT-induced carcinogenesis are not well defined. Mesothelin (MSLN) is overexpressed in many human tumors, including mesotheliomas and pancreatic and ovarian carcinomas. In this study, the role of MSLN in the carcinogenic transformation of human bronchial epithelial cells chronically exposed to single-walled CNT (BSW) was investigated. MSLN overexpression was found in human lung tumors, lung cancer cell lines, and BSW cells. The functional role of MSLN in the BSW cells was then investigated by using stably transfected MSLN knockdown (BSW shMSLN) cells. MSLN knockdown resulted in significantly decreased invasion, migration, colonies on soft agar, and tumor sphere formation. In vivo, BSW shMSLN cells formed smaller primary tumors and less metastases. The mechanism by which MSLN contributes to these more aggressive behaviors was investigated by using ingenuity pathway analysis, which predicted that increased MSLN could induce cyclin E expression. We found that BSW shMSLN cells had decreased cyclin E, and their proliferation rate was reverted to nearly that of untransformed cells. Cell cycle analysis showed that the BSW shMSLN cells had an increased G2 population and a decreased S phase population, which is consistent with the decreased rate of proliferation. Together, our results indicate a novel role of MSLN in the malignant transformation of bronchial epithelial cells following CNT exposure, suggesting its utility as a potential biomarker and drug target for CNT-induced malignancies. PMID:27422997

  15. Staphylococcus aureus α-Toxin Induces Actin Filament Remodeling in Human Airway Epithelial Model Cells.

    PubMed

    Ziesemer, Sabine; Eiffler, Ina; Schönberg, Alfrun; Müller, Christian; Hochgräfe, Falko; Beule, Achim G; Hildebrandt, Jan-Peter

    2018-04-01

    Exposure of cultured human airway epithelial model cells (16HBE14o-, S9) to Staphylococcus aureus α-toxin (hemolysin A, Hla) induces changes in cell morphology and cell layer integrity that are due to the inability of the cells to maintain stable cell-cell or focal contacts and to properly organize their actin cytoskeletons. The aim of this study was to identify Hla-activated signaling pathways involved in regulating the phosphorylation level of the actin-depolymerizing factor cofilin. We used recombinant wild-type hemolysin A (rHla) and a variant of Hla (rHla-H35L) that is unable to form functional transmembrane pores to treat immortalized human airway epithelial cells (16HBE14o-, S9) as well as freshly isolated human nasal tissue. Our results indicate that rHla-mediated changes in cofilin phosphorylation require the formation of functional Hla pores in the host cell membrane. Formation of functional transmembrane pores induced hypophosphorylation of cofilin at Ser3, which was mediated by rHla-induced attenuation of p21-activated protein kinase and LIM kinase activities. Because dephosphorylation of pSer3-cofilin results in activation of this actin-depolymerizing factor, treatment of cells with rHla resulted in loss of actin stress fibers from the cells and destabilization of cell shape followed by the appearance of paracellular gaps in the cell layers. Activation of protein kinase A or activation of small GTPases (Rho, Rac, Cdc42) do not seem to be involved in this response.

  16. The Effect of Commonly Used Excipients on the Epithelial Integrity of Human Cervicovaginal Tissue.

    PubMed

    Hu, Minlu; Zhou, Tian; Dezzutti, Charlene S; Rohan, Lisa C

    Pharmaceutical excipients are widely used in vaginal drug products. The epithelial integrity of the cervicovaginal tissue is important for HIV-1 prevention. However, the effects of excipients on cervicovaginal epithelium remain unknown. This study aims at assessing the effects of vaginal product excipients on the integrity of human cervicovaginal epithelium and on a lead HIV prevention antiretroviral drug, tenofovir (TFV). In the current study, nine excipients commonly used in vaginal formulations were incubated for 6 h with excised human ectocervical tissue. The effects of the excipients were examined by measuring the transepithelial electrical resistance (TEER), epithelial morphology, paracellular/transcellular permeability, and cell viability. The efficacy of TFV for preventing HIV-1 infection in the ex vivo cultured ectocervix was also tested. We found that disodium ethyl-enediaminetetraacetate (EDTA), sorbic acid, and benzoic acid had no effect on the tissue TEER. Butylated hydroxyanisole, glycerin, propylene glycol, methylparaben, and propylparaben slightly to moderately decreased tissue TEER, whereas citric acid significantly decreased the TEER in a time-dependent manner. Tissue morphology observed post-exposure strongly correlated with TEER data; however, a less strong correlation was observed between paracellular permeability and TEER data after exposure to different excipients. In addition, treatment with EDTA, methylparaben, and propylene glycol at tested levels had no effect on the efficacy of TFV in preventing tissue HIV-1 infection. In conclusion, the combined measurements of TEER, morphology, permeability, and viability using human cervicovaginal tissue represent a clinically relevant platform for safety evaluation of excipients and formulated products for HIV-1 prevention.

  17. Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress–mediated apoptosis: possible implication in asthma

    PubMed Central

    Merendino, Anna M.; Paul, Catherine; Vignola, Antonio M.; Costa, Maria A.; Melis, Mario; Chiappara, Giuseppina; Izzo, V.; Bousquet, J.; Arrigo, André-Patrick

    2002-01-01

    Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide–induced apoptosis in human bronchial epithelial 16–HBE cells when they were genetically modified to express reduced levels of Hsp27. PMID:12482203

  18. Treatment of chronic desquamative gingivitis using tissue-engineered human cultured gingival epithelial sheets: a case report.

    PubMed

    Okuda, Kazuhiro; Momose, Manabu; Murata, Masashi; Saito, Yoshinori; lnoie, Masukazu; Shinohara, Chikara; Wolff, Larry F; Yoshie, Hiromasa

    2004-04-01

    Human cultured gingival epithelial sheets were used as an autologous grafting material for regenerating gingival tissue in the maxillary left and mandibular right quadrants of a patient with chronic desquamative gingivitis. Six months post-surgery in both treated areas, there were gains in keratinized gingiva and no signs of gingival inflammation compared to presurgery. In the maxillary left quadrant, preoperative histopathologic findings revealed the epithelium was separated from the connective tissue and inflammatory cells were extensive. After grafting with the gingival epithelial sheets, inflammatory cells were decreased and separation between epithelium and connective tissue was not observed. The human cultured gingival epithelial sheets fabricated using tissue engineering technology showed significant promise for gingival augmentation in periodontal therapy.

  19. Vitamin D3 analog maxacalcitol (OCT) induces hCAP-18/LL-37 production in human oral epithelial cells.

    PubMed

    Tada, Hiroyuki; Shimizu, Takamitsu; Nagaoka, Isao; Takada, Haruhiko

    2016-01-01

    Maxacalcitol (22-oxacalcitriol: OCT) is a synthetic vitamin D3 analog with a limited calcemic effect. In this study, we investigated whether OCT increases the production of LL-37/CAP-18, a human cathelicidin antimicrobial peptide, in human gingival/oral epithelial cells. A human gingival epithelial cell line (Ca9-22) and human oral epithelial cell lines (HSC-2, HSC-3, and HSC-4) exhibited the enhanced expression of LL-37 mRNA upon stimulation with OCT as well as active metabolites of vitamins D3 and D2. Among the human epithelial cell lines, Ca9-22 exhibited the strongest response to these vitamin D-related compounds. OCT induced the higher production of CAP-18 (ng/mL order) until 6 days time-dependently in Ca9-22 cells in culture. The periodontal pathogen Porphyromonas gingivalis was killed by treatment with the LL-37 peptide. These findings suggest that OCT induces the production of hCAP-18/LL-37 in a manner similar to that induced by the active metabolite of vitamin D3.

  20. Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)

    PubMed Central

    Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

    2012-01-01

    Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

  1. Differential effects of human papillomavirus type 6, 16, and 18 DNAs on immortalization and transformation of human cervical epithelial cells

    SciTech Connect

    Pecoraro, G.; Morgan, D.; Defendi, V.

    1989-01-01

    The human papillomaviruses (HPVs) are associated with specific benign and malignant lesions of the skin and mucosal epithelia. Cloned viral DNAs from HPV types 6b, 16, and 18 associated with different pathological manifestations of genital neoplasia in vivo were introduced into primary human cervical epithelial cells by electroporation. Cells transfected with HPV16 or HPV18 DNA acquired indefinite lifespans, distinct morphological alterations, and anchorage-independent growth (HPV18), and contain integrated transcriptionally active viral genomes. HPV6b or plasmid electroporated cells senesced at low passage. The alterations in growth and differentiation of the cells appear to reflect the progressive oncogenic processes that result inmore » cervical carcinoma in vivo.« less

  2. CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential responsiveness of human intestinal epithelial cells to LPS

    PubMed Central

    Dang, Xitong; Eliceiri, Brian P.; Baird, Andrew; Costantini, Todd W.

    2015-01-01

    The human genome contains a unique, distinct, and human-specific α7-nicotinic acetylcholine receptor (α7nAChR) gene [CHRNA7 (gene-encoding α7-nicotinic acetylcholine receptor)] called CHRFAM7A (gene-encoding dup-α7-nicotinic acetylcholine receptor) on a locus of chromosome 15 associated with mental illness, including schizophrenia. Located 5′ upstream from the “wild-type” CHRNA7 gene that is found in other vertebrates, we demonstrate CHRFAM7A expression in a broad range of epithelial cells and sequenced the CHRFAM7A transcript found in normal human fetal small intestine epithelial (FHs) cells to prove its identity. We then compared its expression to CHRNA7 in 11 gut epithelial cell lines, showed that there is a differential response to LPS when compared to CHRNA7, and characterized the CHRFAM7A promoter. We report that both CHRFAM7A and CHRNA7 gene expression are widely distributed in human epithelial cell lines but that the levels of CHRFAM7A gene expression vary up to 5000-fold between different gut epithelial cells. A 3-hour treatment of epithelial cells with 100 ng/ml LPS increased CHRFAM7A gene expression by almost 1000-fold but had little effect on CHRNA7 gene expression. Mapping the regulatory elements responsible for CHRFAM7A gene expression identifies a 1 kb sequence in the UTR of the CHRFAM7A gene that is modulated by LPS. Taken together, these data establish the presence, identity, and differential regulation of the human-specific CHRFAM7A gene in human gut epithelial cells. In light of the fact that CHRFAM7A expression is reported to modulate ligand binding to, and alter the activity of, the wild-type α7nAChR ligand-gated pentameric ion channel, the findings point to the existence of a species-specific α7nAChR response that might regulate gut epithelial function in a human-specific fashion.—Dang, X., Eliceiri, B. P., Baird, A., Costantini, T. W. CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential

  3. Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells

    PubMed Central

    Emam, Aufaugh; Carter, William G; Lingwood, Clifford

    2010-01-01

    Internalization of PAK strain Pseudomonas aeruginosa into human respiratory epithelial cell lines and HeLa cervical cancer cells in vitro was readily demonstrable via a gentamycin protection assay. Depletion of target cell glycosphingolipids (GSLs) using a glucosyl ceramide synthase inhibitor, P4, completely prevented P. aeruginosa internalization. In contrast, P4 treatment had no effect on the internalization of Salmonella typhimurium into HeLa cells. Internalized P. aeruginosa were within membrane vacuoles, often containing microvesicles, between the bacterium and the limiting membrane. P. aeruginosa internalization was markedly enhanced by target cell pretreatment with the exogenous GSL, deacetyl gangliotetraosyl ceramide (Gg4). Gg4 binds the lipid raft marker, GM1 ganglioside. Target cell pretreatment with TLCK, but not other (serine) protease inhibitors, prevented both P. aeruginosa host cell binding and internalization. NFkB inhibition also prevented internalization. A GSL-containing lipid-raft model of P. aeruginosa host cell binding/internalization is proposed PMID:21270937

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

    NASA Technical Reports Server (NTRS)

    Applewhite, Lisa C.

    2002-01-01

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

  5. Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

    NASA Astrophysics Data System (ADS)

    Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

    2015-06-01

    The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

  6. Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Han, Meng; Blindewald-Wittich, Almut; Holz, Frank G.; Giese, Günter; Niemz, Markolf H.; Snyder, Sarah; Sun, Hui; Yu, Jiayi; Agopov, Michael; La Schiazza, Olivier; Bille, Josef F.

    2006-01-01

    Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events that trigger the death of RPE cells at the subcellular level. Two-photon excited autofluorescence (TPEF) imaging of RPE cells proves to be well suited to investigate both the morphological and the spectral characteristics of the human RPE cells. The dominant fluorophores of autofluorescence derive from lipofuscin (LF) granules that accumulate in the cytoplasm of the RPE cells with increasing age. Spectral TPEF imaging reveals the existence of abnormal LF granules with blue shifted autofluorescence in RPE cells of aging patients and brings new insights into the complicated composition of the LF granules. Based on a proposed two-photon laser scanning ophthalmoscope, TPEF imaging of the living retina may be valuable for diagnostic and pathological studies of age related eye diseases.

  7. Photodynamic actions of indocyanine green and trypan blue on human lens epithelial cells in vitro.

    PubMed

    Melendez, Robert F; Kumar, Neeru; Maswadi, Saher M; Zaslow, Kenneth; Glickmank, Randolph D

    2005-07-01

    The purpose of this study was to evaluate the toxicity and photodynamic activity of indocyanine green (ICG) and trypan blue (TryB) on cultured human lensepithelial cells (LECs). Experimental study. Lens epithelial cell viability was assessed after treatment with ICG and TryB concentrations ranging from 0.025 to 5.0 mg/ml, and exposure to 806 nm diode laser. At ICG concentrations below 0.5 mg/ml, there was > or =75% cell viability; at higher ICG concentrations there was dose-dependent cytotoxicity in addition to loss of cellular viability due to ICG photosensitization. TryB had little cytotoxicity to the LECs: >80% cells were viable irrespective of the dye concentration or laser treatment. These data indicate that ICG may have application as a photosensitizer in the selective eradication of residual LECs after cataract surgery to reduce the incidence of posterior capsule opacification.

  8. Productive Lifecycle of Human Papillomaviruses that Depends Upon Squamous Epithelial Differentiation

    PubMed Central

    Kajitani, Naoko; Satsuka, Ayano; Kawate, Akifumi; Sakai, Hiroyuki

    2012-01-01

    Human papillomaviruses (HPVs) target the stratified epidermis, and can causes diseases ranging from benign condylomas to malignant tumors. Infections of HPVs in the genital tract are among the most common sexually transmitted diseases, and a major risk factor for cervical cancer. The virus targets epithelial cells in the basal layer of the epithelium, while progeny virions egress from terminally differentiated cells in the cornified layer, the surface layer of the epithelium. In infected basal cells, the virus maintains its genomic DNA at low-copy numbers, at which the viral productive lifecycle cannot proceed. Progression of the productive lifecycle requires differentiation of the host cell, indicating that there is tight crosstalk between viral replication and host differentiation programs. In this review, we discuss the regulation of the HPV lifecycle controlled by the differentiation program of the host cells. PMID:22536200

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

    DOEpatents

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

    1983-01-01

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

  10. Protective effects of Lactobacillus plantarum against epithelial barrier dysfunction of human colon cell line NCM460

    PubMed Central

    Liu, Zhi-Hua; Shen, Tong-Yi; Zhang, Peng; Ma, Yan-Lei; Moyer, Mary Pat; Qin, Huan-Long

    2010-01-01

    AIM: To investigate the effects of Lactobacillus plantarum (L. plantarum) in the intestinal permeability and expression of tight junction (TJ) using the normal human colon cell line NCM460. METHODS: Paracellular permeability of NCM460 monolayers was determined by transepithelial electrical resistance and dextran permeability. Expression of TJ proteins in NCM460 cell monolayers was detected by Western blotting and quantitative real-time polymerase chain reaction. RESULTS: L. plantarum played an important role in increasing transepithelial electrical resistance and decreasing the permeability to macromolecules of NCM460 monolayers against the disruption caused by enteropathogenic Escherichia coli (E. coli) or enteroinvasive E. coli. L. plantarum also prevented the decrease in the expression of TJ proteins and F-actin in NCM460 cells. CONCLUSION: L. plantarum can protect against dysfunction of NCM460 intestinal epithelial barrier caused by enteropathogenic E. coli or enteroinvasive E. coli, and thus can be a potential candidate of therapeutic agents for the treatment of intestinal diseases. PMID:21128328

  11. Isolation and purification of ILS, an interleukin 1 inhibitor produced by human gingival epithelial cells.

    PubMed Central

    Walsh, L J; Lander, P E; Seymour, G J; Powell, R N

    1987-01-01

    Recent studies have described the presence of an interleukin 1 (IL-1) inhibitor (ILS) in supernatants from human gingival organ cultures. This report describes the isolation and purification of ILS. ILS was produced in serum-free medium and purified to apparent homogeneity by ion exchange chromatography and high pressure liquid chromatography. IL-1 and ILS eluted as discrete proteins using this system, with ILS appearing as a single protein band of 97,400 molecular weight on SDS-PAGE. ILS inhibited the effects of both murine IL-1 and IL-2 on thymocyte proliferation, and was heat resistant, moderately resistant to freeze-thawing and stable for 2 years at 0-4 degrees C or -20 degrees C. Production of ILS by gingival epithelial cell cultures was not affected by depletion of Langerhans cells, implying that ILS is primarily a keratinocyte product. Images Fig. 5 PMID:3498574

  12. Emergence of fractal geometry on the surface of human cervical epithelial cells during progression towards cancer

    NASA Astrophysics Data System (ADS)

    Dokukin, M. E.; Guz, N. V.; Woodworth, C. D.; Sokolov, I.

    2015-03-01

    Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation.

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

    NASA Technical Reports Server (NTRS)

    Richmond, Robert C.

    2001-01-01

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

  14. Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells.

    PubMed

    Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

    2015-06-12

    The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

  15. Emerging of fractal geometry on surface of human cervical epithelial cells during progression towards cancer

    PubMed Central

    Dokukin, M. E.; Guz, N. V.; Woodworth, C.D.; Sokolov, I.

    2015-01-01

    Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation. PMID:25844044

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

    PubMed

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

    2006-01-01

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

  17. Mucin Characteristics of Human Corneal-Limbal Epithelial Cells that Exclude the Rose Bengal Anionic Dye

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2015-01-22

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

  19. Cellular and molecular alterations in human epithelial cells transformed by high let radiation

    NASA Astrophysics Data System (ADS)

    Hei, T. K.; Piao, C. Q.; Sutter, T.; Willey, J. C.; Suzuki, K.

    An understanding of the radiobiological effects of high LET radiation is essential for human risk estimation and radiation protection. In the present study, we show that a single, 30 cGy dose of 150 keV/mum ^4He ions can malignantly transform human papillomavirus immortalized human bronchial epithelial [BEP2D] cells. Transformed cells produce progressively growing tumors in nude mice. The transformation frequency by the single dose of alpha particles is estimated to be approximately 4 x 10^-7. Based on the average cross-sectional area of BEP2D cells, it can be calculated that a mean traversal of 1.4 particles per cell is sufficient to induce tumorigenic conversion of these cells 3 to 4 months post-irradiation. Tumorigenic BEP2D cells overexpress mutated p53 tumor suppressor oncoproteins in addition to the cell cycle control gene cyclin D1 and D2. This model provides an opportunity to study the cellular and molecular changes at the various stages in radiation carcinogenesis involving human cells.

  20. Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

    SciTech Connect

    Hirano, Seishiro, E-mail: seishiro@nies.go.j; Fujitani, Yuji; Furuyama, Akiko

    Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC{sub 50} of MWCNT was 12 {mu}g/ml, whereas that of asbestos (crocidolite) was 678 {mu}g/ml. Overmore » the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 {mu}g/ml. BEAS-2B cells were exposed to 2, 5, or 10 {mu}g/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-{kappa}B or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-{kappa}B was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-{kappa}B, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.« less

  1. Hypoxia induces mucin expression and secretion in human bronchial epithelial cells.

    PubMed

    Zhou, Xiangdong; Tu, Jing; Li, Qi; Kolosov, Victor P; Perelman, Juliy M

    2012-12-01

    The study objective was to investigate the role of hypoxia-inducible factor 1 (HIF-1) in the transcriptional activation of MUC5AC in human bronchial epithelial (HBE) 16 cells under hypoxia conditions and the effect of hypoxia on expression and secretion of MUC5AC. Cells were incubated in hypoxia medium. Serial deletions or mutations of the MUC5AC promoter were cloned in the reporter pGL3-basic plasmid (Promega Biotech Co, Ltd, Beijing, China). These reporter plasmids were cotransfected with HIF-1α small interfering RNA. Hypoxia markedly increased the level of MUC5AC secretion and the transcriptional activity of MUC5AC promoters. Western blot analysis showed that HIF-1α and MUC5AC proteins were strongly increased after HBE16 cells were exposed to hypoxic conditions. Treatment of HBE16 cells with HIF-1α inhibitor (YC-1) or HIF-1α small interfering RNA significantly inhibited the expression of HIF-1α and MUC5AC, and the secretion of MUC5AC. Depletion of the promoter sequence did not reduce the MUC5AC promoter activity to hypoxia. Luciferase assay indicated that HRE in the MUC5AC promoter was in the region from -120 to +54. Promoter sequence analysis showed that 1 HRE site at -65 plays an important role in hypoxia activation of the MUC5AC. The inactivation of the HRE site using site-directed mutagenesis led to the complete loss of induction by hypoxia, which further confirmed the key role of the HRE site. MUC5AC expression and secretion are upregulated in response to hypoxia. The HRE site at -65 in the MUC5AC promoter and the HIF-1α are the major regulators for the cellular response against hypoxia in human bronchial epithelial cells. Copyright © 2012 Mosby, Inc. All rights reserved.

  2. The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

    PubMed

    Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

    2004-09-01

    Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

  3. High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells

    PubMed Central

    Ye, J; Wu, H; Wu, Y; Wang, C; Zhang, H; Shi, X; Yang, J

    2012-01-01

    Purpose To investigate the toxic effects of ethylenediaminetetraacetic acid disodium salt (EDTA), a corneal penetration enhancer in topical ophthalmic formulations, on DNA in human corneal epithelial cells (HCEs), and to investigate whether the effect induced by EDTA can be inhibited by high molecular weight hyaluronan (HA). Methods Cells were exposed to EDTA in concentrations ranging from 0.00001 to 0.01% for 60 min, or 30 min high molecular weight HA pretreatment followed by EDTA treatment. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. The DNA single- and double-strand breaks of HCEs were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci, respectively. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2′, 7′-dichlorodihydrofluorescein diacetate. Results EDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in human corneal epithelial cells at concentrations lower than 0.01%. However, a significant increase of DNA single- and double-strand breaks was observed in a dose-dependent manner with all the concentrations of EDTA tested in HCEs. In addition, EDTA treatment led to elevated ROS generation. Moreover, 30 min preincubation with high molecular weight HA significantly decreased EDTA-induced ROS generation and DNA damage. Conclusions EDTA could induce DNA damage in HCEs, probably through oxidative stress. Furthermore, high molecular weight HA was an effective protective agent that had antioxidant properties and decreased DNA damage induced by EDTA. PMID:22595911

  4. Oxidative stress mediated cytotoxicity of biologically synthesized silver nanoparticles in human lung epithelial adenocarcinoma cell line

    PubMed Central

    2014-01-01

    The goal of the present study was to investigate the toxicity of biologically prepared small size of silver nanoparticles in human lung epithelial adenocarcinoma cells A549. Herein, we describe a facile method for the synthesis of silver nanoparticles by treating the supernatant from a culture of Escherichia coli with silver nitrate. The formation of silver nanoparticles was characterized using various analytical techniques. The results from UV-visible (UV-vis) spectroscopy and X-ray diffraction analysis show a characteristic strong resonance centered at 420 nm and a single crystalline nature, respectively. Fourier transform infrared spectroscopy confirmed the possible bio-molecules responsible for the reduction of silver from silver nitrate into nanoparticles. The particle size analyzer and transmission electron microscopy results suggest that silver nanoparticles are spherical in shape with an average diameter of 15 nm. The results derived from in vitro studies showed a concentration-dependent decrease in cell viability when A549 cells were exposed to silver nanoparticles. This decrease in cell viability corresponded to increased leakage of lactate dehydrogenase (LDH), increased intracellular reactive oxygen species generation (ROS), and decreased mitochondrial transmembrane potential (MTP). Furthermore, uptake and intracellular localization of silver nanoparticles were observed and were accompanied by accumulation of autophagosomes and autolysosomes in A549 cells. The results indicate that silver nanoparticles play a significant role in apoptosis. Interestingly, biologically synthesized silver nanoparticles showed more potent cytotoxicity at the concentrations tested compared to that shown by chemically synthesized silver nanoparticles. Therefore, our results demonstrated that human lung epithelial A549 cells could provide a valuable model to assess the cytotoxicity of silver nanoparticles. PMID:25242904

  5. Effects of bile acids on human airway epithelial cells: implications for aerodigestive diseases.

    PubMed

    Aldhahrani, Adil; Verdon, Bernard; Ward, Chris; Pearson, Jeffery

    2017-01-01

    Gastro-oesophageal reflux and aspiration have been associated with chronic and end-stage lung disease and with allograft injury following lung transplantation. This raises the possibility that bile acids may cause lung injury by damaging airway epithelium. The aim of this study was to investigate the effect of bile acid challenge using the immortalised human bronchial epithelial cell line (BEAS-2B). The immortalised human bronchial epithelial cell line (BEAS-2B) was cultured. A 48-h challenge evaluated the effect of individual primary and secondary bile acids. Post-challenge concentrations of interleukin (IL)-8, IL-6 and granulocyte-macrophage colony-stimulating factor were measured using commercial ELISA kits. The viability of the BEAS-2B cells was measured using CellTiter-Blue and MTT assays. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid were successfully used to stimulate cultured BEAS-2B cells at different concentrations. A concentration of lithocholic acid above 10 μmol·L -1 causes cell death, whereas deoxycholic acid, chenodeoxycholic acid and cholic acid above 30 μmol·L -1 was required for cell death. Challenge with bile acids at physiological levels also led to a significant increase in the release of IL-8 and IL6 from BEAS-2B. Aspiration of bile acids could potentially cause cell damage, cell death and inflammation in vivo . This is relevant to an integrated gastrointestinal and lung physiological paradigm of chronic lung disease, where reflux and aspiration are described in both chronic lung diseases and allograft injury.

  6. Oxidative stress mediated cytotoxicity of biologically synthesized silver nanoparticles in human lung epithelial adenocarcinoma cell line

    NASA Astrophysics Data System (ADS)

    Han, Jae Woong; Gurunathan, Sangiliyandi; Jeong, Jae-Kyo; Choi, Yun-Jung; Kwon, Deug-Nam; Park, Jin-Ki; Kim, Jin-Hoi

    2014-09-01

    The goal of the present study was to investigate the toxicity of biologically prepared small size of silver nanoparticles in human lung epithelial adenocarcinoma cells A549. Herein, we describe a facile method for the synthesis of silver nanoparticles by treating the supernatant from a culture of Escherichia coli with silver nitrate . The formation of silver nanoparticles was characterized using various analytical techniques. The results from UV-visible (UV-vis) spectroscopy and X-ray diffraction analysis show a characteristic strong resonance centered at 420 nm and a single crystalline nature, respectively. Fourier transform infrared spectroscopy confirmed the possible bio-molecules responsible for the reduction of silver from silver nitrate into nanoparticles. The particle size analyzer and transmission electron microscopy results suggest that silver nanoparticles are spherical in shape with an average diameter of 15 nm. The results derived from in vitro studies showed a concentration-dependent decrease in cell viability when A549 cells were exposed to silver nanoparticles. This decrease in cell viability corresponded to increased leakage of lactate dehydrogenase (LDH), increased intracellular reactive oxygen species generation (ROS), and decreased mitochondrial transmembrane potential (MTP). Furthermore, uptake and intracellular localization of silver nanoparticles were observed and were accompanied by accumulation of autophagosomes and autolysosomes in A549 cells. The results indicate that silver nanoparticles play a significant role in apoptosis. Interestingly, biologically synthesized silver nanoparticles showed more potent cytotoxicity at the concentrations tested compared to that shown by chemically synthesized silver nanoparticles. Therefore, our results demonstrated that human lung epithelial A549 cells could provide a valuable model to assess the cytotoxicity of silver nanoparticles.

  7. Biodiesel exhaust-induced cytotoxicity and proinflammatory mediator production in human airway epithelial cells.

    PubMed

    Mullins, Benjamin J; Kicic, Anthony; Ling, Kak-Ming; Mead-Hunter, Ryan; Larcombe, Alexander N

    2016-01-01

    Increasing use of biodiesel has prompted research into the potential health effects of biodiesel exhaust exposure. Few studies directly compare the health consequences of mineral diesel, biodiesel, or blend exhaust exposures. Here, we exposed human epithelial cell cultures to diluted exhaust generated by the combustion of Australian ultralow-sulfur-diesel (ULSD), unprocessed canola oil, 100% canola biodiesel (B100), and a blend of 20% canola biodiesel mixed with 80% ULSD. The physicochemical characteristics of the exhaust were assessed and we compared cellular viability, apoptosis, and levels of interleukin (IL)-6, IL-8, and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) in exposed cultured cells. Different fuel types produced significantly different amounts of exhaust gases and different particle characteristics. All exposures resulted in significant apoptosis and loss of viability when compared with control, with an increasing proportion of biodiesel being correlated with a decrease in viability. In most cases, exposure to exhaust resulted in an increase in mediator production, with the greatest increases most often in response to B100. Exposure to pure canola oil (PCO) exhaust did not increase mediator production, but resulted in a significant decrease in IL-8 and RANTES in some cases. Our results show that canola biodiesel exhaust exposure elicits inflammation and reduces viability of human epithelial cell cultures in vitro when compared with ULSD exhaust exposure. This may be related to an increase in particle surface area and number in B100 exhaust when compared with ULSD exhaust. Exposure to PCO exhaust elicited the greatest loss of cellular viability, but virtually no inflammatory response, likely due to an overall increase in average particle size. © 2014 Wiley Periodicals, Inc.

  8. Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation.

    PubMed

    Zhao, Bin; Zhang, Yijie; Han, Shichao; Zhang, Wei; Zhou, Qin; Guan, Hao; Liu, Jiaqi; Shi, Jihong; Su, Linlin; Hu, Dahai

    2017-04-01

    Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.

  9. Immunohistochemical localisation of keratin and luminal epithelial antigen in myoepithelial and luminal epithelial cells of human mammary and salivary gland tumours.

    PubMed

    Nathrath, W B; Wilson, P D; Trejdosiewicz, L K

    1982-01-01

    Rabbit antisera to human 40-63 000 MW epidermal keratin, one batch with restricted distribution of reactivity from an initial (aK1) and one with "broad spectrum" distribution of reactivity from a late bleeding (aK), and to "luminal epithelial antigen" (aLEA) were applied to formalin fixed paraffin embedded sections of human normal and neoplastic mammary and salivary glands using an indirect immunoperoxidase method. aK1 reacted with myoepithelial cells, aLEA with luminal epithelial cells and aK with both cell types in normal mammary and salivary gland. In breast carcinomas the majority of intraluminal and infiltrating carcinoma cells reacted with aLEA but not with aK1 which reacted only with surrounding myoepithelial cells. aK reacted with both myoepithelial cells and with intraluminal and infiltrating tumour cells. In the salivary gland adenomas the majority of cells reacted with aK, and those cells arranged in a tubular fashion reacted with aLEA.

  10. Upregulation of SQSTM1/p62 contributes to nickel-induced malignant transformation of human bronchial epithelial cells.

    PubMed

    Huang, Haishan; Zhu, Junlan; Li, Yang; Zhang, Liping; Gu, Jiayan; Xie, Qipeng; Jin, Honglei; Che, Xun; Li, Jingxia; Huang, Chao; Chen, Lung-Chi; Lyu, Jianxin; Gao, Jimin; Huang, Chuanshu

    2016-10-02

    Chronic lung inflammation is accepted as being associated with the development of lung cancer caused by nickel exposure. Therefore, identifying the molecular mechanisms that lead to a nickel-induced sustained inflammatory microenvironment that causes transformation of human bronchial epithelial cells is of high significance. In the current studies, we identified SQSTM1/p62 as a novel nickel-upregulated protein that is important for nickel-induced inflammatory TNF expression, subsequently resulting in transformation of human bronchial epithelial cells. We found that nickel exposure induced SQSTM1 protein upregulation in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The SQSTM1 upregulation was also observed in human lung squamous cell carcinoma. Further studies revealed that the knockdown of SQSTM1 expression dramatically inhibited transformation of human lung epithelial cells upon chronic nickel exposure, whereas ectopic expression of SQSTM1 promoted such transformation. Mechanistic studies showed that the SQSTM1 upregulation by nickel was the compromised result of upregulating SQSTM1 mRNA transcription and promoting SQSTM1 protein degradation. We demonstrated that nickel-initiated SQSTM1 protein degradation is mediated by macroautophagy/autophagy via an MTOR-ULK1-BECN1 axis, whereas RELA is important for SQSTM1 transcriptional upregulation following nickel exposure. Furthermore, SQSTM1 upregulation exhibited its promotion of nickel-induced cell transformation through exerting an impetus for nickel-induced inflammatory TNF mRNA stability. Consistently, the MTOR-ULK1-BECN1 autophagic cascade acted as an inhibitory effect on nickel-induced TNF expression and cell transformation. Collectively, our results demonstrate a novel SQSTM1 regulatory network that promotes a nickel-induced tumorigenic effect in human bronchial epithelial cells, which is negatively controlled by an autophagic cascade following nickel exposure.

  11. Upregulation of SQSTM1/p62 contributes to nickel-induced malignant transformation of human bronchial epithelial cells

    PubMed Central

    Huang, Haishan; Zhu, Junlan; Li, Yang; Zhang, Liping; Gu, Jiayan; Xie, Qipeng; Jin, Honglei; Che, Xun; Li, Jingxia; Huang, Chao; Chen, Lung-Chi; Lyu, Jianxin; Gao, Jimin; Huang, Chuanshu

    2016-01-01

    ABSTRACT Chronic lung inflammation is accepted as being associated with the development of lung cancer caused by nickel exposure. Therefore, identifying the molecular mechanisms that lead to a nickel-induced sustained inflammatory microenvironment that causes transformation of human bronchial epithelial cells is of high significance. In the current studies, we identified SQSTM1/p62 as a novel nickel-upregulated protein that is important for nickel-induced inflammatory TNF expression, subsequently resulting in transformation of human bronchial epithelial cells. We found that nickel exposure induced SQSTM1 protein upregulation in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The SQSTM1 upregulation was also observed in human lung squamous cell carcinoma. Further studies revealed that the knockdown of SQSTM1 expression dramatically inhibited transformation of human lung epithelial cells upon chronic nickel exposure, whereas ectopic expression of SQSTM1 promoted such transformation. Mechanistic studies showed that the SQSTM1 upregulation by nickel was the compromised result of upregulating SQSTM1 mRNA transcription and promoting SQSTM1 protein degradation. We demonstrated that nickel-initiated SQSTM1 protein degradation is mediated by macroautophagy/autophagy via an MTOR-ULK1-BECN1 axis, whereas RELA is important for SQSTM1 transcriptional upregulation following nickel exposure. Furthermore, SQSTM1 upregulation exhibited its promotion of nickel-induced cell transformation through exerting an impetus for nickel-induced inflammatory TNF mRNA stability. Consistently, the MTOR-ULK1-BECN1 autophagic cascade acted as an inhibitory effect on nickel-induced TNF expression and cell transformation. Collectively, our results demonstrate a novel SQSTM1 regulatory network that promotes a nickel-induced tumorigenic effect in human bronchial epithelial cells, which is negatively controlled by an autophagic cascade following nickel exposure. PMID:27467530

  12. INHIBITION OF RESPIRATORY SYNCYTIAL VIRUS (RSV)-INDUCED INFLAMMATION BY 3-NITROTYROSINE IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Inhibition of Respiratory Syncytial Virus (RSV)-Induced Inflammation by 3-Nitrotyrosine in Human Bronchial Epithelial Cells. J. M. Soukup, MPH 1, ZW. Li, MD 2 and YC. T. Huang, MD 1. 1 NHEERL, US Environmental Protection Agency, RTP, NC and 2 CEMALB, University of North Carolina,...

  13. ULTRAFINE CARBON PARTICLES INDUCE IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS THROUGH A POST-TRANSCRIPTIONAL MECHANISM

    EPA Science Inventory

    Ultrafine carbon particles induce IL-8 expression in human airway
    epithelial cells through a post-transcritpional mechanism
    Epidemiological studies suggest that ultrafine particles contribute to
    particulate matter (PM) - induced adverse health effects. IL-8 is an
    i...

  14. INCREASED IL-8 AND IL-6 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    INCREASED IL-6 AND IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES.
    R Silbajoris1, A G Lenz2, I Jaspers3, J M Samet1. 1NHEERL, USEPA, RTP, NC, USA; 2GSF-Institute for Inhalation Biology, Neuherberg, Germany; 3 CEMLB, UNC-CH, Chapel Hill, ...

  15. Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal-Limbal and Human Conjunctival Epithelial Cells.

    PubMed

    Schuerer, Nadine; Stein, Elisabeth; Inic-Kanada, Aleksandra; Pucher, Marion; Hohenadl, Christine; Bintner, Nora; Ghasemian, Ehsan; Montanaro, Jacqueline; Barisani-Asenbauer, Talin

    2017-06-01

    To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal-limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT-based assay. The morphology of cells was also investigated. HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface.

  16. Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal–Limbal and Human Conjunctival Epithelial Cells

    PubMed Central

    Schuerer, Nadine; Stein, Elisabeth; Inic-Kanada, Aleksandra; Pucher, Marion; Hohenadl, Christine; Bintner, Nora; Ghasemian, Ehsan; Montanaro, Jacqueline

    2017-01-01

    Purpose: To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal–limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. Methods: HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT–based assay. The morphology of cells was also investigated. Results: HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. Conclusions: We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface. PMID:28399036

  17. Cigarette smoke extract modulates human beta-defensin-2 and interleukin-8 expression in human gingival epithelial cells.

    PubMed

    Mahanonda, R; Sa-Ard-Iam, N; Eksomtramate, M; Rerkyen, P; Phairat, B; Schaecher, K E; Fukuda, M M; Pichyangkul, S

    2009-08-01

    Human gingival epithelial cells (HGECs) are continually exposed to oral bacteria and to other harmful agents. Their responses to stimuli are critical in maintaining periodontal homeostasis. The aim of this study was to investigate the modulating effect of cigarette smoke extract (CSE) on the innate immune responses of HGECs. Toll-like receptor (TLR) expression of HGECs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effect of CSE or nicotine on the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) and the pro-inflammatory cytokine interleukin (IL)-8 in stimulated HGEC cultures was evaluated by RT-PCR and enzyme-linked immunosorbent assay. The HGECs expressed mRNA of TLRs 1, 2, 3, 5, 6, 9, 10, and minimally of TLR4, but not of TLRs 7 or 8. Stimulation of HGECs with highly purified TLR2, 3 or 5 ligands led to expression of hBD-2 and of IL-8. Enhancement of hBD-2 and IL-8 was observed in HGECs after combined stimulation with Porphyromonas gingivalis lipopolysaccharide (TLR2 ligand) and tumour necrosis factor-alpha, compared with stimulation using either agent alone. After CSE exposure, hBD-2 expression was markedly reduced in stimulated HGEC cultures, whereas IL-8 expression was markedly increased. These effects were also observed, but were markedly attenuated, upon nicotine treatment. Human gingival epithelial cells play a critical role in orchestrating the innate immune responses of periodontal tissue via TLR signalling. Our results represent the first demonstration that CSE can modulate HGEC function by suppressing hBD-2 and enhancing IL-8 production, and this may be, in part, a possible mechanism which promotes periodontal disease.

  18. Human Milk Oligosaccharides Attenuate Antigen-Antibody Complex Induced Chemokine Release from Human Intestinal Epithelial Cell Lines.

    PubMed

    Zehra, Sehrish; Khambati, Ibrahim; Vierhout, Megan; Mian, M Firoz; Buck, Rachael; Forsythe, Paul

    2018-02-01

    There has been increased interest in the use of dietary ingredients, including prebiotics such as human-milk oligosaccharides (HMOs), as therapeutic strategies for food allergy. Understanding the mechanisms underlying the beneficial effects of HMOs is important to realizing their therapeutic potential. Here we demonstrate that the HMO, 6'-sialyllactose (6'SL) inhibited chemokine (IL-8 and CCL20) release from T-84 and HT-29 cells stimulated with antigen-antibody complex, TNFα or PGE 2 ; an effect that was PPARγ dependent and associated with decreased activity of the transcription factors AP-1 and NFκB. In contrast, 2'-fucosyllactose (2'FL) selectively inhibited CCL20 release in response to antigen antibody complex in a PPARγ independent manner. This study reinforces the concept that structurally different oligosaccharides have distinct biological activities and identifies, for the first time, that the HMOs, 6'SL, and 2'FL, modulate human epithelial cell responses related to allergic disease. These findings encourage further investigation of the therapeutic potential of specific HMOs in food allergy. This study provides evidence for direct effects of HMOs in addition to their prebiotic role and demonstrates, for the first time, modulation of Ag-IgE complex activation of human epithelial cells that may have important implications for food-allergy. The study also reinforces the concept that structurally different oligosaccharides have distinct biological activities. In determining the composition of infant formula, addition of oligosaccharides with specific structures may provide direct modulation of immune responses and potentially attenuate symptoms or development of food allergy. © 2018 Institute of Food Technologists®.

  19. Interferon-gamma increased epithelial barrier function via upregulating claudin-7 expression in human submandibular gland duct epithelium.

    PubMed

    Abe, Ayumi; Takano, Kenichi; Kojima, Takashi; Nomura, Kazuaki; Kakuki, Takuya; Kaneko, Yakuto; Yamamoto, Motohisa; Takahashi, Hiroki; Himi, Tetsuo

    2016-06-01

    Tight junctions (TJs) are necessary for salivary gland function and may serve as indicators of salivary gland epithelial dysfunction. IgG4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition which disrupts the TJ associated epithelial barrier. The salivary glands are one of the most frequently involved organs in IgG4-RD, however, changes of the TJ associated epithelial barrier in salivary gland duct epithelium is poorly understood. Here, we investigated the regulation and function of TJs in human submandibular gland ductal epithelial cells (HSDECs) in normal and IgG4-RD. We examined submandibular gland (SMG) tissue from eight control individuals and 22 patients with IgG4-RD and established an HSDEC culture system. Immunohistochemistry, immunocytochemistry, western blotting, and measurement of transepithelial electrical resistance (TER) were performed. Claudin-4, claudin-7, occludin, and JAM-A were expressed at the apical side of the duct epithelium in submandibular gland (SMG) tissue and at the cell borders in HSDECs of normal and IgG4-RD. The expression and distribution of TJs in SMG tissue were not different in control individuals and patients with IgG4-RD in vivo and in vitro. Although interferon-gamma (IFNγ) generally disrupts the integrity and function of TJs, as manifested by decreased epithelial barrier function, IFNγ markedly increased the epithelial barrier function of HSDECs via upregulation of claudin-7 expression in HSDECs from patients with IgG4-RD. This is the first report showing an IFNγ-dependent increase in epithelial barrier function in the salivary gland duct epithelium. Our results provide insights into the functional significance of TJs in salivary gland duct epithelium in physiological and pathological conditions, including IgG4-RD.

  20. BCL-2 Modifying Factor (BMF) Is a Central Regulator of Anoikis in Human Intestinal Epithelial Cells*

    PubMed Central

    Hausmann, Martin; Leucht, Katharina; Ploner, Christian; Kiessling, Stephan; Villunger, Andreas; Becker, Helen; Hofmann, Claudia; Falk, Werner; Krebs, Michaela; Kellermeier, Silvia; Fried, Michael; Schölmerich, Jürgen; Obermeier, Florian; Rogler, Gerhard

    2011-01-01

    BCL-2 modifying factor (BMF) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma 2. B-cell lymphoma 2 is neutralized by BMF and thereby facilitates cytochrome C release from mitochondria. We investigated the role of BMF for intestinal epithelial cell (IEC) homeostasis. Acute colitis was induced in Bmf-deficient mice (Bmf−/−) with dextran sulfate sodium. Colonic crypt length in Bmf−/− mice was significantly increased as compared with WT mice. Dextran sulfate sodium induced less signs of colitis in Bmf−/− mice, as weight loss was reduced compared with the WT. Primary human IEC exhibited increased BMF in the extrusion zone. Quantitative PCR showed a significant up-regulation of BMF expression after initiation of anoikis in primary human IEC. BMF was found on mitochondria during anoikis, as demonstrated by Western blot analysis. RNAi mediated knockdown of BMF reduced the number of apoptotic cells and led to reduced caspase 3 activity. A significant increase in phospho-AKT was determined after RNAi treatment. BMF knockdown supports survival of IEC. BMF is induced in human IEC by the loss of cell attachment and is likely to play an important role in the regulation of IEC survival. PMID:21673109

  1. Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies

    PubMed Central

    2011-01-01

    Micro-RNAs (miRs) are important regulators of mRNA and protein expression; the ability of miR expression profilings to distinguish different cancer types and classify their sub-types has been well-described. They also represent a novel biological entity with potential value as tumour biomarkers, which can improve diagnosis, prognosis, and monitoring of treatment response for human cancers. This endeavour has been greatly facilitated by the stability of miRs in formalin-fixed paraffin-embedded (FFPE) tissues, and their detection in circulation. This review will summarize some of the key dysregulated miRs described to date in human epithelial malignancies, and their potential value as molecular bio-markers in FFPE tissues and blood samples. There remain many challenges in this domain, however, with the evolution of different platforms, the complexities of normalizing miR profiling data, and the importance of evaluating sufficiently-powered training and validation cohorts. Nonetheless, well-conducted miR profiling studies should contribute important insights into the molecular aberrations driving human cancer development and progression. PMID:22128797

  2. Effect of guaifenesin on mucin production, rheology, and mucociliary transport in differentiated human airway epithelial cells.

    PubMed

    Seagrave, JeanClare; Albrecht, Helmut; Park, Yong Sung; Rubin, Bruce; Solomon, Gail; Kim, K Chul

    2011-12-01

    Guaifenesin is widely used to alleviate symptoms of excessive mucus accumulation in the respiratory tract. However, its mechanism of action is poorly understood. The authors hypothesized that guaifenesin improves mucociliary clearance in humans by reducing mucin release, by decreasing mucus viscoelasticity, and by increasing mucociliary transport. To test these hypotheses, human differentiated airway epithelial cells, cultured at an air-liquid interface, were treated with clinically relevant concentrations of guaifenesin by addition to the basolateral medium. To evaluate the effect on mucin secretion, the authors used an anzyme-linked immunosorbent assay (ELISA) to measure the amounts of MUC5AC protein in apical surface fluid and cell lysates. To measure mucociliary transportability, additional cultures were treated for 1 or 6 hours with guaifenesin, and the movement of cell debris was measured from video data. Further, the authors measured mucus dynamic viscoelasticity using a micro cone and plate rheometer with nondestructive creep transformation. Guaifenesin suppressed mucin production in a dose-dependent manner at clinically relevant concentrations. The reduced mucin production was associated with increased mucociliary transport and decreased viscoelasticity of the mucus. Viability of the cultures was not significantly affected. These results suggest that guaifenesin could improve mucociliary clearance in humans by reducing the release and/or production of mucins, thereby altering mucus rheology.

  3. An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

    PubMed Central

    Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. Steven; Qian, Wei-Jun

    2010-01-01

    Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell line by applying anti-phosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that the majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems. PMID:19534553

  4. Establishment of a blue light damage model of human retinal pigment epithelial cells in vitro.

    PubMed

    Su, G; Cai, S J; Gong, X; Wang, L L; Li, H H; Wang, L M

    2016-06-24

    To establish a blue-light damage model of human retinal pigment epithelium (RPE). Fourth-generation human RPE cells were randomly divided into two groups. In group A, cells were exposed to blue light (2000 ± 500 lux) for 0 (control), 3, 6, 9, and 12 h, and cell culture was stopped after 12 h. In group B, cells were exposed to blue light at the same intensity and time periods, but cell culture was stopped after 24 h. TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed to determine the most suitable illuminating time with apoptotic index. Flow cytometry was used to determine apoptotic ratio of RPEs. In group A, the apoptotic index of cells that received 6, 9 and 12 h of blue light was higher than that of control. The apoptotic index of cells receiving 9 and 12 h was higher than that of 6 h (P = 0.000). In group B, the apoptotic index and RPE cell apoptosis ratio of cells exposed to 6, 9 and 12 h of blue light were higher than that of 3 h (P = 0.000); and cells receiving 9 and 12 h had higher values than that of 6 h. This study demonstrated that the best conditions to establish a blue light damage model of human retinal pigment epithelial cells in vitro are 2000 ± 500 lux light intensity for 6 h, with 24 h of cell culture post-exposure.

  5. Effects of polysaccharide isolated from Streptococcus thermophilus CRL1190 on human gastric epithelial cells.

    PubMed

    Marcial, Guillermo; Messing, Jutta; Menchicchi, Bianca; Goycoolea, Francisco M; Faller, Gerhard; Graciela, Font de Valdez; Hensel, Andreas

    2013-11-01

    EPS1190 was isolated from skim milk fermented with Stretococcus thermophilus CRL1190. The polysaccharide consisted of 33% glucose and 66% galactose with 1,4- and 1,4,6-galactose residues as main building blocks beside a high amount of 1,4-linked glucose. The polymer was characterized additionally concerning viscosity and zeta potential. EPS1190 stimulated cellular vitality and proliferation of human stomach AGS cells and human buccal KB cells significantly. EPS1190 stimulated phagocytosis rate of murine macrophages RAW264.7 significantly. NO-release or anti-inflammatory effects by inhibition of LPS-induced NO release were not observed. Confocal laser scanning microscopy revealed that EPS1190 is partially internalized into AGS cells via endosomes. The bioadhesive absorption of FITC-labeled EPS1190 into the mucus layer on the apical side of the epithelium using histological tissue sections from human stomach was observed. Specific interaction of EPS1190 with mucin can be excluded as shown by microviscosimetry studies. EPS1190 increased the adhesion of H. pylori to AGS cells, which resulted in increased secretion of proinflammatory cytokines TNFa, IL-6 and IL-8. Summarizing, EPS1190 seems to stimulate epithelial cell regeneration and immunological innate defense mechanisms, which again can rationalized the use of this polysaccharide as cytoprotective compound in probiotioc preparations. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Stimulation of GPR30 Increases Release of EMMPRIN-Containing Microvesicles in Human Uterine Epithelial Cells

    PubMed Central

    Light, Mallory M.; Mehrotra, Pavni; Nowak, Romana A.

    2012-01-01

    Context: Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. Objective: The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). Design: We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. Results: We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. Conclusions: These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology. PMID:23012390

  7. Stimulation of GPR30 increases release of EMMPRIN-containing microvesicles in human uterine epithelial cells.

    PubMed

    Burnett, Lindsey A; Light, Mallory M; Mehrotra, Pavni; Nowak, Romana A

    2012-12-01

    Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology.

  8. The species translation challenge—A systems biology perspective on human and rat bronchial epithelial cells

    PubMed Central

    Poussin, Carine; Mathis, Carole; Alexopoulos, Leonidas G; Messinis, Dimitris E; Dulize, Rémi H J; Belcastro, Vincenzo; Melas, Ioannis N; Sakellaropoulos, Theodore; Rhrissorrakrai, Kahn; Bilal, Erhan; Meyer, Pablo; Talikka, Marja; Boué, Stéphanie; Norel, Raquel; Rice, John J; Stolovitzky, Gustavo; Ivanov, Nikolai V; Peitsch, Manuel C; Hoeng, Julia

    2014-01-01

    The biological responses to external cues such as drugs, chemicals, viruses and hormones, is an essential question in biomedicine and in the field of toxicology, and cannot be easily studied in humans. Thus, biomedical research has continuously relied on animal models for studying the impact of these compounds and attempted to ‘translate’ the results to humans. In this context, the SBV IMPROVER (Systems Biology Verification for Industrial Methodology for PROcess VErification in Research) collaborative initiative, which uses crowd-sourcing techniques to address fundamental questions in systems biology, invited scientists to deploy their own computational methodologies to make predictions on species translatability. A multi-layer systems biology dataset was generated that was comprised of phosphoproteomics, transcriptomics and cytokine data derived from normal human (NHBE) and rat (NRBE) bronchial epithelial cells exposed in parallel to more than 50 different stimuli under identical conditions. The present manuscript describes in detail the experimental settings, generation, processing and quality control analysis of the multi-layer omics dataset accessible in public repositories for further intra- and inter-species translation studies. PMID:25977767

  9. The species translation challenge-a systems biology perspective on human and rat bronchial epithelial cells.

    PubMed

    Poussin, Carine; Mathis, Carole; Alexopoulos, Leonidas G; Messinis, Dimitris E; Dulize, Rémi H J; Belcastro, Vincenzo; Melas, Ioannis N; Sakellaropoulos, Theodore; Rhrissorrakrai, Kahn; Bilal, Erhan; Meyer, Pablo; Talikka, Marja; Boué, Stéphanie; Norel, Raquel; Rice, John J; Stolovitzky, Gustavo; Ivanov, Nikolai V; Peitsch, Manuel C; Hoeng, Julia

    2014-01-01

    The biological responses to external cues such as drugs, chemicals, viruses and hormones, is an essential question in biomedicine and in the field of toxicology, and cannot be easily studied in humans. Thus, biomedical research has continuously relied on animal models for studying the impact of these compounds and attempted to 'translate' the results to humans. In this context, the SBV IMPROVER (Systems Biology Verification for Industrial Methodology for PROcess VErification in Research) collaborative initiative, which uses crowd-sourcing techniques to address fundamental questions in systems biology, invited scientists to deploy their own computational methodologies to make predictions on species translatability. A multi-layer systems biology dataset was generated that was comprised of phosphoproteomics, transcriptomics and cytokine data derived from normal human (NHBE) and rat (NRBE) bronchial epithelial cells exposed in parallel to more than 50 different stimuli under identical conditions. The present manuscript describes in detail the experimental settings, generation, processing and quality control analysis of the multi-layer omics dataset accessible in public repositories for further intra- and inter-species translation studies.

  10. Syndecan-1 suppresses epithelial-mesenchymal transition and migration in human oral cancer cells.

    PubMed

    Wang, Xiaofeng; He, Jinting; Zhao, Xiaoming; Qi, Tianyang; Zhang, Tianfu; Kong, Chenfei

    2018-04-01

    Epithelial-mesenchymal transition (EMT) is one of the major processes that contribute to the occurrence of cancer metastasis. EMT has been associated with the development of oral cancer. Syndecan‑1 (SDC1) is a key cell‑surface adhesion molecule and its expression level inversely correlates with tumor differentiation and prognosis. In the present study, we aimed to determine the role of SDC1 in oral cancer progression and investigate the molecular mechanisms through which SDC1 regulates the EMT and invasiveness of oral cancer cells. We demonstrated that basal SDC1 expression levels were lower in four oral cancer cell lines (KB, Tca8113, ACC2 and CAL‑27), than in normal human periodontal ligament fibroblasts. Ectopic overexpression of SDC1 resulted in morphological transformation, decreased expression of EMT‑associated markers, as well as decreased migration, invasiveness and proliferation of oral cancer cells. In contrast, downregulation of the expression of SDC1 caused the opposite results. Furthermore, the knockdown of endogenous SDC1 activated the extracellular signal‑regulated kinase (ERK) cascade, upregulated the expression of Snail and inhibited the expression of E‑cadherin. In conclusion, our findings revealed that SDC1 suppressed EMT via the modulation of the ERK signaling pathway that, in turn, negatively affected the invasiveness of human oral cancer cells. Our results provided useful evidence about the potential use of SDC1 as a molecular target for therapeutic interventions in human oral cancer.

  11. Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

    PubMed

    Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

    2003-09-01

    Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the

  12. [The effect of UV-irradiation on telomerase activity and other stress-related proteins in human lens epithelial cells].

    PubMed

    Wu, Zhi-hong; Zhang, Jin-song

    2005-05-01

    To investigate the changes and the role of telomerase activity and other stress-related proteins in the process of UV-induced DNA damage and repair in human lens epithelial cells. Human lens epithelial cells were irradiated at UV-doses 0.0 (control group) and 0.5, 1.5, 2.5, 3.5, 5.0, 7.5, 10.0 mJ/cm(2) (treated 1-7 group). Telomerase activity was determined by Telomerase Repeat Amplification Protocol-Enzyme Linked Immunosorbent Assay (TRAP-ELISA), p53, growth arrest and DNA damage inducible (GADD45), proliferating cell nuclear antigen (PCNA) and p16 protein levels were analyzed by Western blotting. Telomerase activity in control group and treated 1-7 group showed increased tendency, the differences of telomerase activity in 8 groups were significantly (P < 0.01). The expression of p53, GADD45, PCNA, p16 proteins showed increased tendency in experimental group, comparing with the control group, there were significant difference (P < 0.01). During UV-induced DNA damage and repair in human lens epithelial cells, telomerase activity was upregulated and the expression of stress-related proteins levels was increased. Upregulated telomerase activity may play both a protective and a proliferative role in human lens epithelial cells. Increased stress-related proteins level is critic in UV-induced DNA damage and repair in human lens epithelial. Increased telomerase activity is associated with increased levels of the stress-related proteins.

  13. Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells.

    PubMed

    Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X

    2004-08-01

    To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. Bacterial infection of HCECs induces

  14. Role of EGFR Transactivation in Preventing Apoptosis in Pseudomonas aeruginosa–Infected Human Corneal Epithelial Cells

    PubMed Central

    Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X.

    2009-01-01

    PURPOSE To determine the role of epidermal growth factor (EGF) receptor (EGFR)–mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa–infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase–mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis

  15. Calcium dependent and independent cytokine synthesis by air pollution particle-exposed human bronchial epithelial cells

    SciTech Connect

    Sakamoto, Noriho; Hayashi, Shizu; Gosselink, John

    2007-12-01

    Exposure to ambient air pollution particles with a diameter of < 10 {mu}m (PM{sub 10}) has been associated with increased cardiopulmonary morbidity and mortality. We have shown that human bronchial epithelial cells (HBECs) exposed to PM{sub 10} produce pro-inflammatory mediators that contribute to a local and systemic inflammatory response. Changes in intracellular calcium concentrations ([Ca{sup 2+}]{sub i}) have been demonstrated to regulate several functions of the airway epithelium including the production of pro-inflammatory mediators. The aim of the present study was to determine the nature and mechanism of calcium responses induced by PM{sub 10} in HBECs and its relationship tomore » cytokine synthesis. Methods: Primary HBECs were exposed to urban air pollution particles (EHC-93) and [Ca{sup 2+}]{sub i} responses were measured using the fluoroprobe (Fura-2). Cytokine levels were measured at mRNA and protein levels using real-time PCR and ELISA. Results: PM{sub 10} increased [Ca{sup 2+}]{sub i} in a dose-dependent manner. This calcium response was reduced by blocking the influx of calcium into cells (i.e. calcium-free medium, NiCl{sub 2}, LaCl{sub 3}). PM{sub 10} also decreased the activity of calcium pumps. PM{sub 10} increased the production of IL-1{beta}, IL-8, GM-CSF and LIF. Preincubation with intracellular calcium chelator (BAPTA-AM) attenuated IL-1{beta} and IL-8 production, but not GM-CSF and LIF production. Conclusion: We conclude that exposure to PM{sub 10} induces an increase in cytosolic calcium and cytokine production in bronchial epithelial cells. Our results also suggest that PM{sub 10} induces the production of pro-inflammatory mediators via either intracellular calcium-dependent (IL-1{beta}, IL-8) or -independent (GM-CSF, LIF) pathways.« less

  16. Antiandrogenic actions of medroxyprogesterone acetate on epithelial cells within normal human breast tissues cultured ex vivo.

    PubMed

    Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E

    2014-01-01

    Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

  17. Effect of budesonide and azelastine on histamine signaling regulation in human nasal epithelial cells.

    PubMed

    Liu, Shao-Cheng; Lin, Chun-Shu; Chen, Shyi-Gen; Chu, Yueng-Hsiang; Lee, Fei-Peng; Lu, Hsuan-Hsuan; Wang, Hsing-Won

    2017-02-01

    Both glucocorticoids and H1-antihistamines are widely used on patients with airway diseases. However, their direct effects on airway epithelial cells are not fully explored. Therefore, we use the primary culture of human nasal epithelial cells (HNEpC) to delineate in vitro mucosal responses to above two drugs. HNEpC cells were cultured with/without budesonide and azelastine. The growth rate at each group was recorded and measured as population double time (PDT). The histamine1-receptor (H1R), muscarinic1-receptor (M1R) and M3R were measured using immunocytochemistry and western blotting after 7-days treatment. Then, we used histamine and methacholine to stimulate the mucus secretion from HNEpC and observed the MUC5AC expression in culture supernatants. Concentration-dependent treatment-induced inhibition of HNEpC growth rate was observed. Cells incubated with azelastine proliferated significantly slower than that with budesonide and the combined use of those drugs led to significant PDT prolong. The immunocytochemistry showed the H1R, M1R and M3R were obviously located in the cell membrane without apparent difference after treatment. However, western blotting showed that budesonide can significantly up-regulate the H1R, M1R and M3R level while azelastine had opposite effects. Histamine and methacholine stimulated MUC5AC secretion was greater in cells treated with budesonide but was lesser in those treated with azelastine, as compared to controls. Our data suggest that both budesonide and azelastine can significantly inhibit HNEpC proliferation, and therefore, be helpful in against airway remodeling. Long-term use of budesonide might amplify histamine signaling and result in airway hyperreactivity to stimulants by enhancing H1R, M1R and M3R expression while azelastine can oppose this effect. Therefore, combined use of those two drugs in patients with chronic inflammatory airway diseases may be an ideal option.

  18. E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells

    PubMed Central

    Aug, Argo; Altraja, Siiri; Kilk, Kalle; Porosk, Rando; Soomets, Ursel; Altraja, Alan

    2015-01-01

    E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1’s maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes. PMID:26536230

  19. E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells.

    PubMed

    Aug, Argo; Altraja, Siiri; Kilk, Kalle; Porosk, Rando; Soomets, Ursel; Altraja, Alan

    2015-01-01

    E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1's maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.

  20. Proteomic Signatures of Human Oral Epithelial Cells in HIV-Infected Subjects

    PubMed Central

    Yohannes, Elizabeth; Ghosh, Santosh K.; Jiang, Bin; McCormick, Thomas S.; Weinberg, Aaron; Hill, Edward; Faddoul, Faddy; Chance, Mark R.

    2011-01-01

    The oral epithelium, the most abundant structural tissue lining the oral mucosa, is an important line of defense against infectious microorganisms. HIV infected subjects on highly active antiretroviral therapy (HAART) are susceptible to comorbid viral, bacterial and fungal infections in the oral cavity. To provide an assessment of the molecular alterations of oral epithelia potentially associated with susceptibility to comorbid infections in such subjects, we performed various proteomic studies on over twenty HIV infected and healthy subjects. In a discovery phase two Dimensional Difference Gel Electrophoresis (2-D DIGE) analyses of human oral gingival epithelial cell (HOEC) lysates were carried out; this identified 61 differentially expressed proteins between HIV-infected on HAART subjects and healthy controls. Down regulated proteins in HIV-infected subjects include proteins associated with maintenance of protein folding and pro- and anti-inflammatory responses (e.g., heat-shock proteins, Cryab, Calr, IL-1RA, and Galectin-3-binding protein) as well as proteins involved in redox homeostasis and detoxification (e.g., Gstp1, Prdx1, and Ero1). Up regulated proteins include: protein disulfide isomerases, proteins whose expression is negatively regulated by Hsp90 (e.g., Ndrg1), and proteins that maintain cellular integrity (e.g., Vimentin). In a verification phase, proteins identified in the protein profiling experiments and those inferred from Ingenuity Pathway Analysis were analyzed using Western blotting analysis on separate HOEC lysate samples, confirming many of the discovery findings. Additionally in HIV-infected patient samples Heat Shock Factor 1 is down regulated, which explains the reduced heat shock responses, while activation of the MAPK signal transduction cascade is observed. Overall, HAART therapy provides an incomplete immune recovery of the oral epithelial cells of the oral cavity for HIV-infected subjects, and the toxic side effects of HAART and

  1. Nerve growth factor reduces amiloride‐sensitive Na+ transport in human airway epithelial cells

    PubMed Central

    Shimko, Michael J.; Zaccone, Eric J.; Thompson, Janet A.; Schwegler‐Berry, Diane; Kashon, Michael L.; Fedan, Jeffrey S.

    2014-01-01

    Abstract Nerve growth factor (NGF) is overexpressed in patients with inflammatory lung diseases, including virus infections. Airway surface liquid (ASL), which is regulated by epithelial cell ion transport, is essential for normal lung function. No information is available regarding the effect of NGF on ion transport of airway epithelium. To investigate whether NGF can affect ion transport, human primary air‐interface cultured epithelial cells were placed in Ussing chambers to obtain transepithelial voltage (−7.1 ± 3.4 mV), short‐circuit current (Isc, 5.9 ± 1.0 μA), and transepithelial resistance (750 Ω·cm2), and to measure responses to ion transport inhibitors. Amiloride (apical, 3.5 × 10−5 mol/L) decreased Isc by 55.3%. Apically applied NGF (1 ng/mL) reduced Isc by 5.3% in 5 min; basolaterally applied NGF had no effect. The response to amiloride was reduced (41.6%) in the presence of NGF. K‐252a (10 nmol/L, apical) did not itself affect Na+ transport, but it attenuated the NGF‐induced reduction in Na+ transport, indicating the participation of the trkA receptor in the NGF‐induced reduction in Na+ transport. PD‐98059 (30 μmol/L, apical and basolateral) did not itself affect Na+ transport, but attenuated the NGF‐induced reduction in Na+ transport, indicating that trkA activated the Erk 1/2 signaling cascade. NGF stimulated phosphorylation of Erk 1/2 and the β‐subunit of ENaC. K‐252a and PD‐98059 inhibited these responses. NGF had no effect on Isc in the presence of apical nystatin (50 μmol/L). These results indicate that NGF inhibits Na+ transport through a trkA‐Erk 1/2‐activated signaling pathway linked to ENaC phosphorylation. PMID:25347857

  2. Candidate Microbicides Block HIV-1 Infection of Human Immature Langerhans Cells within Epithelial Tissue Explants

    PubMed Central

    Kawamura, Tatsuyoshi; Cohen, Sandra S.; Borris, Debra L.; Aquilino, Elisabeth A.; Glushakova, Svetlana; Margolis, Leonid B.; Orenstein, Jan M.; Offord, Robin E.; Neurath, A. Robert; Blauvelt, Andrew

    2000-01-01

    Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1Ba-L infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08–4.77%). HIV-1–infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1–1 μg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1Ba-L (an R5 HIV-1 strain) more efficiently infected LC–T cell cocultures when compared with HIV-1IIIB (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1. PMID:11085750

  3. Studies on the bioavailability of the provitamin A carotenoid, beta-carotene, using human exfoliated colonic epithelial cells.

    PubMed

    Gireesh, T; Nair, P P; Sudhakaran, P R

    2004-08-01

    The possibility of using exfoliated colonic epithelial cells for assessing the bioavailability of beta-carotene was examined. Analysis of exfoliated colonic epithelial cells showed the presence of beta-carotene and vitamin A. The beta-carotene content was significantly lower in cells from stool samples of subjects on a beta-carotene-poor diet than those receiving a single dose of a beta-carotene supplement. Colonic epithelial cells isolated from stool samples collected daily during a wash-out period while the subjects were on a beta-carotene-poor diet showed a steady decrease in beta-carotene content, reaching the lowest value on day 7. Kinetic analysis showed that a single dose of a beta-carotene supplement in the form of spirulina (Spirulina platensis) or agathi (Sesbania grandiflora) after the wash-out period caused an increase in the beta-carotene content after a lag period of 5-7 d, but the vitamin A levels during these periods were not significantly affected. Analysis of plasma beta-carotene concentration also showed similar changes, which correlated with those of exfoliated colonic cells. A relationship between the beta-carotene content of the diet and that of the colonic epithelial cells suggests that analysis of the beta-carotene content in exfoliated human colonic epithelial cells is a useful non-invasive method to assess the bioavailability of provitamin A beta-carotene.

  4. The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.

    PubMed Central

    Halbert, C L; Demers, G W; Galloway, D A

    1992-01-01

    Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation. Images PMID:1312623

  5. Separation of malignant human breast cancer epithelial cells from healthy epithelial cells using an advanced dielectrophoresis-activated cell sorter (DACS).

    PubMed

    An, Jaemin; Lee, Jangwon; Lee, Sang Ho; Park, Jungyul; Kim, Byungkyu

    2009-06-01

    In this paper, we successfully separated malignant human breast cancer epithelial cells (MCF 7) from healthy breast cells (MCF 10A) and analyzed the main parameters that influence the separation efficiency with an advanced dielectrophoresis (DEP)-activated cell sorter (DACS). Using the efficient DACS, the malignant cancer cells (MCF 7) were isolated successfully by noninvasive methods from normal cells with similar cell size distributions (MCF 10A), depending on differences between their material properties such as conductivity and permittivity, because our system was able to discern the subtle differences in the properties by generating continuously changed electrical field gradients. In order to evaluate the separation performance without considering size variations, the cells collected from each outlet were divided into size-dependent groups and counted statistically. Following that, the quantitative relative ratio of numbers between MCF 7 and MCF 10A cells in each size-dependent group separated by the DEP were compared according to applied frequencies in the range 48, 51, and 53 MHz with an applied amplitude of 8 V(pp). Finally, under the applied voltage of 48 MHz-8 V(pp) and a flow rate of 290 microm/s, MCF 7 and MCF 10A cells were separated with a maximum efficiency of 86.67% and 98.73% respectively. Therefore, our suggested system shows it can be used for detection and separation of cancerous epithelial cells from noncancerous cells in clinical applications.

  6. Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells.

    PubMed

    Balder, Rachel; Lipski, Serena; Lazarus, John J; Grose, William; Wooten, Ronald M; Hogan, Robert J; Woods, Donald E; Lafontaine, Eric R

    2010-09-28

    Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms. Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells) and A549 (type II pneumocytes), as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures.A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705). The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to thrive inside J774A.1 murine macrophages

  7. Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation

    NASA Astrophysics Data System (ADS)

    Armitage, Mark

    Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or

  8. Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis

    PubMed Central

    Teoh-Fitzgerald, ML; Fitzgerald, MP; Zhong, W; Askeland, RW; Domann, FE

    2013-01-01

    Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging

  9. Differential gene expression profiling of functionally and developmentally distinct human prostate epithelial populations.

    PubMed

    Liu, Haibo; Cadaneanu, Radu M; Lai, Kevin; Zhang, Baohui; Huo, Lihong; An, Dong Sun; Li, Xinmin; Lewis, Michael S; Garraway, Isla P

    2015-05-01

    Human fetal prostate buds appear in the 10th gestational week as solid cords, which branch and form lumens in response to androgen 1. Previous in vivo analysis of prostate epithelia isolated from benign prostatectomy specimens indicated that Epcam⁺ CD44⁻ CD49f(Hi) basal cells possess efficient tubule initiation capability relative to other subpopulations 2. Stromal interactions and branching morphogenesis displayed by adult tubule-initiating cells (TIC) are reminiscent of fetal prostate development. In the current study, we evaluated in vivo tubule initiation by human fetal prostate cells and determined expression profiles of fetal and adult epithelial subpopulations in an effort to identify pathways used by TIC. Immunostaining and FACS analysis based on Epcam, CD44, and CD49f expression demonstrated the majority (99.9%) of fetal prostate epithelial cells (FC) were Epcam⁺ CD44⁻ with variable levels of CD49f expression. Fetal populations isolated via cell sorting were implanted into immunocompromised mice. Total RNA isolation from Epcam⁺ CD44⁻ CD49f(Hi) FC, adult Epcam⁺ CD44⁻ CD49f(Hi) TIC, Epcam⁺ CD44⁺ CD49f(Hi) basal cells (BC), and Epcam⁺ CD44⁻ CD49f(Lo) luminal cells (LC) was performed, followed by microarray analysis of 19 samples using the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data was analyzed using Partek Genomics Suite Version 6.4. Genes selected showed >2-fold difference in expression and P < 5.00E-2. Results were validated with RT-PCR. Grafts retrieved from Epcam⁺ CD44⁻ fetal cell implants displayed tubule formation with differentiation into basal and luminal compartments, while only stromal outgrowths were recovered from Epcam- fetal cell implants. Hierarchical clustering revealed four distinct groups determined by antigenic profile (TIC, BC, LC) and developmental stage (FC). TIC and BC displayed basal gene expression profiles, while LC expressed secretory genes. FC had a unique profile with the most similarities

  10. Human vaginal epithelial cells augment autophagy marker genes in response to Candida albicans infection.

    PubMed

    Shroff, Ankit; Sequeira, Roicy; Reddy, Kudumula Venkata Rami

    2017-04-01

    Autophagy plays an important role in clearance of intracellular pathogens. However, no information is available on its involvement in vaginal infections such as vulvo-vaginal candidiasis (VVC). VVC is intimately associated with the immune status of the human vaginal epithelial cells (VECs). The objective of our study is to decipher if autophagy process is involved during Candida albicans infection of VECs. In this study, C. albicans infection system was established using human VEC line (VK2/E6E7). Infection-induced change in the expression of autophagy markers like LC3 and LAMP-1 were analyzed by RT-PCR, q-PCR, Western blot, immunofluorescence and transmission electron microscopy (TEM) studies were carried out to ascertain the localization of autophagosomes. Multiplex ELISA was carried out to determine the cytokine profiles. Analysis of LC3 and LAMP-1 expression at mRNA and protein levels at different time points revealed up-regulation of these markers 6 hours post C. albicans infection. LC3 and LAMP-1 puncti were observed in infected VECs after 12 hours. TEM studies showed C. albicans entrapped in autophagosomes. Cytokines-TNF-α and IL-1β were up-regulated in culture supernatants of VECs at 12 hours post-infection. The results suggest that C. albicans invasion led to the activation of autophagy as a host defense mechanism of VECs. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. An immunohistochemical survey of pS2 expression in human epithelial cancers.

    PubMed

    Luqmani, Y A; Ryall, G; Shousha, S; Coombes, R C

    1992-01-21

    The oestrogen-inducible pS2 protein isolated from human breast-cancer cells appears to have prognostic significance in breast-cancer patients. Using both monoclonal and polyclonal pS2 anti-sera, we have carried out an immunocytochemical survey of 9 epithelial cancers. Some degree of specific tumour staining was observed in 4 tissues, the extent varying greatly between specimens. Most (11/13) of the colorectal cancers showed immunoreactivity, as did 4/5 pancreatic carcinomas. In some of these cases, patchy staining was also observed in adjacent non-tumour cells. Two bronchio-alveolar carcinomas showed positivity, but 5 poorly differentiated adenocarcinomas of the lung did not. In the ovary, staining was observed in 3/3 mucinous cystadenocarcinomas but only in 1/8 of the serous type. No reactivity was seen in specimens of salivary gland, kidney, liver, prostate or uterus. This pilot study indicates that pS2 may be a useful marker of adenocarcinomas in human neoplasms apart from those in the breast, and suggests that more extensive surveys might be worthwhile.

  12. Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

    NASA Astrophysics Data System (ADS)

    Hei, T. K.; Piao, C. Q.; Wu, L. J.; Willey, J. C.; Hall, E. J.

    1998-11-01

    Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon 56Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to 56Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.

  13. On physical changes on surface of human cervical epithelial cells during cancer transformations

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor; Dokukin, Maxim; Guz, Nataliia; Woodworth, Craig

    2013-03-01

    Physical changes of the cell surface of cells during transformation from normal to cancerous state are rather poorly studied. Here we describe our recent studies of such changes done on human cervical epithelial cells during their transformation from normal through infected with human papillomavirus type-16 (HPV-16), immortalized (precancerous), to cancerous cells. The changes were studied with the help of atomic force microscopy (AFM) and through the measurement of physical adhesion of fluorescent silica beads to the cell surface. Based on the adhesion experiments, we clearly see the difference in nonspecific adhesion which occurs at the stage of immortalization of cells, precancerous cells. The analysis done with the help of AFM shows that the difference observed comes presumably from the alteration of the cellular ``brush,'' a layer that surrounds cells and which consists of mostly microvilli, microridges, and glycocalyx. Further AFM analysis reveals the emergence of fractal scaling behavior on the surface of cells when normal cells turn into cancerous. The possible causes and potential significance of these observations will be discussed.

  14. Proteomic analysis of secreted proteins by human bronchial epithelial cells in response to cadmium toxicity.

    PubMed

    Chen, De-Ju; Xu, Yan-Ming; Zheng, Wei; Huang, Dong-Yang; Wong, Wing-Yan; Tai, William Chi-Shing; Cho, Yong-Yeon; Lau, Andy T Y

    2015-09-01

    For years, many studies have been conducted to investigate the intracellular response of cells challenged with toxic metal(s), yet, the corresponding secretome responses, especially in human lung cells, are largely unexplored. Here, we provide a secretome analysis of human bronchial epithelial cells (BEAS-2B) treated with cadmium chloride (CdCl2 ), with the aim of identifying secreted proteins in response to Cd toxicity. Proteins from control and spent media were separated by two-dimensional electrophoresis and visualized by silver staining. Differentially-secreted proteins were identified by MALDI-TOF-MS analysis and database searching. We characterized, for the first time, the extracellular proteome changes of BEAS-2B dosed with Cd. Our results unveiled that Cd treatment led to the marked upregulation of molecular chaperones, antioxidant enzymes, enzymes associated with glutathione metabolic process, proteins involved in cellular energy metabolism, as well as tumor-suppressors. Pretreatment of cells with the thiol antioxidant glutathione before Cd treatment effectively abrogated the secretion of these proteins and prevented cell death. Taken together, our results demonstrate that Cd causes oxidative stress-induced cytotoxicity; and the differentially-secreted protein signatures could be considered as targets for potential use as extracellular biomarkers upon Cd exposure. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Specific binding of Haemophilus influenzae to minor gangliosides of human respiratory epithelial cells.

    PubMed Central

    Fakih, M G; Murphy, T F; Pattoli, M A; Berenson, C S

    1997-01-01

    Gangliosides are sialylated glycosphingolipids that serve as receptors for various bacteria. To investigate endogenous gangliosides of human respiratory epithelial cells as potential receptors for Haemophilus influenzae, three strains, including nontypeable H. influenzae (NTHI) 1479, and isogenic fimbriated (f+) and nonfimbriated (f0) H. influenzae type b 770235, were 3H labeled and overlaid on two-dimensional thin-layer chromatography (TLC) plates containing either purified HEp-2 gangliosides or murine brain gangliosides. NTHI 1479 bound exclusively to two distinct minor ganglioside doublets, with mobilities near that of GM1. These minor gangliosides comprised only 14.2 and 9.4% of the total, respectively. NTHI 1479 also bound to a distinct ganglioside of human macrophages whose chromatographic mobilities closely resemble those of one of the NTHI-binding gangliosides of HEp-2 cells. H. influenzae type b 770235 f+ and f0 each bound to a different minor HEp-2 ganglioside doublet, with proportionately weaker affinity for a major ganglioside doublet. Remarkably, none of the three strains bound to any murine brain gangliosides. Moreover, when 80 to 90% of sialic acid residues were enzymatically removed from HEp-2 gangliosides, NTHI 1479 binding was proportionately impaired, compared with untreated controls. Our findings support a role for specific gangliosides of specific cells as receptors for H. influenzae strains. Our findings further demonstrate that individual minor gangliosides possess unique biological properties. PMID:9125549

  16. Adherence of Clostridium perfringens spores to human intestinal epithelial Caco-2 cells.

    PubMed

    Sakanoue, Hideyo; Nakano, Takashi; Sano, Kouichi; Yasugi, Mayo; Monma, Chie; Miyake, Masami

    2018-03-01

    Clostridium perfringens is a gram-positive, spore-forming bacillus, and is a causative agent of foodborne infection, antibiotic-associated diarrhoea and sporadic diarrhoea in humans. In cases of antibiotic-associated and sporadic diarrhoea, C. perfringens colonises the intestine, proliferates and causes disease. However, bacterial colonisation of the intestine is not considered necessary in the pathogenesis of foodborne illness, because such pathogenesis can be explained by anchorage-independent production of diarrhoeic toxin by the bacterium in the intestine. In this study, we used an in vitro adherence assay to examine the adherence of C. perfringens spores to human intestinal Caco-2 cells. Adherence of spores from isolates of foodborne illness and nosocomial infection was observed within 15 min, and plateaued 60 min after inoculation. Electron microscopy revealed a tight association of spores with the surface of Caco-2 cells. The adherence of vegetative cells could not be confirmed by the same method, however. These results suggest that C. perfringens spores may adhere to intestinal epithelial cells in vivo, although its biological significance remains to be determined.

  17. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution.

    PubMed

    Hu, Wen-Yang; Hu, Dan-Ping; Xie, Lishi; Li, Ye; Majumdar, Shyama; Nonn, Larisa; Hu, Hong; Shioda, Toshi; Prins, Gail S

    2017-08-01

    Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Quantitative proteomic analysis of human breast epithelial cells with differential telomere length

    SciTech Connect

    Yu, Li-Rong; Chan, King C.; Tahara, Hidetoshi

    Telomeres play important functional roles in cell proliferation, cell cycle regulation, and genetic stability, in which telomere length is critical. In this study, quantitative proteome comparisons for the human breast epithelial cells with short and long telomeres (184-hTERT{sub L} vs. 184-hTERT{sub S} and 90P-hTERT{sub L} vs. 90P-hTERT{sub S}), resulting from transfection of the human telomerase reverse transcriptase (hTERT) gene, were performed using cleavable isotope-coded affinity tags. More than 2000 proteins were quantified in each comparative experiment, with approximately 77% of the proteins identified in both analyses. In the cells with long telomeres, significant and consistent alterations were observed in metabolismmore » (amino acid, nucleotide, and lipid metabolism), genetic information transmission (transcription and translation regulation, spliceosome and ribosome complexes), and cell signaling. Interestingly, the DNA excision repair pathway is enhanced, while integrin and its ligands are downregulated in the cells with long telomeres. These results may provide valuable information related to telomere functions.« less

  19. Biological Function of Ribosomal Protein L10 on Cell Behavior in Human Epithelial Ovarian Cancer

    PubMed Central

    Shi, Jimin; Zhang, Lingyun; Zhou, Daibing; Zhang, Jinguo; Lin, Qunbo; Guan, Wencai; Zhang, Jihong; Ren, Weimin; Xu, Guoxiong

    2018-01-01

    Ribosomal protein L10 (RPL10) is one of large ribosomal proteins and plays a role in Wilms' tumor and premature ovarian failure. However, the function of RPL10 in human epithelial ovarian cancer (EOC) remains unknown. The purpose of this study was to examine the expression level and function of RPL10 in EOC. RPL10 protein expression was detected by immunohistochemistry and Western blot. The association RPL10 expression with clinical features was analyzed. Loss-of-function and gain-of-function approaches were applied in cellular assays, including cell viability, migration, invasion, and apoptosis. Our study demonstrated for the first time that RPL10 was upregulated in human EOC compared with normal ovarian tissues. Knockdown of RPL10 inhibited cell viability, migration, and invasion, and increased cell apoptosis. On the contrary, upregulation of RPL10 increased cell viability, migration, invasion, and decreased cell apoptosis. Furthermore, miR-143-3p regulated RPL10 expression. Our data indicate that RPL10 is a potential tissue biomarker of patients with EOC and may be a therapeutic target of ovarian cancer. PMID:29556332

  20. HER2 induces expression of leptin in human breast epithelial cells.

    PubMed

    Cha, Yujin; Kang, Youjin; Moon, Aree

    2012-12-01

    A close association between the obesity hormone leptin and breast cancer progression has been suggested. The present study investigated the molecular mechanism for enhanced leptin expression in breast cancer cells and its functional significance in breast cancer aggressiveness. We examined whether leptin expression level is affected by the oncoprotein human epidermal growth factor receptor2 (HER2), which is overexpressed in ∼30% of breast tumors. Here, we report, for the first time, that HER2 induces transcriptional activation of leptin in MCF10A human breast epithelial cells. We also showed that p38 mitogen-activated protein kinase signaling was involved in leptin expression induced by HER2. We showed a crucial role of leptin in the invasiveness of HER2-MCF10A cells using an siRNA molecule targeting leptin. Taken together, the results indicate a molecular link between HER2 and leptin, providing supporting evidence that leptin represents a target for breast cancer therapy. [BMB Reports 2012; 45(12): 719-723].

  1. HER2 induces expression of leptin in human breast epithelial cells

    PubMed Central

    Cha, Yujin; Kang, Youjin; Moon, Aree

    2012-01-01

    A close association between the obesity hormone leptin and breast cancer progression has been suggested. The present study investigated the molecular mechanism for enhanced leptin expression in breast cancer cells and its functional significance in breast cancer aggressiveness. We examined whether leptin expression level is affected by the oncoprotein human epidermal growth factor receptor2 (HER2), which is overexpressed in ∼30% of breast tumors. Here, we report, for the first time, that HER2 induces transcriptional activation of leptin in MCF10A human breast epithelial cells. We also showed that p38 mitogen-activated protein kinase signaling was involved in leptin expression induced by HER2. We showed a crucial role of leptin in the invasiveness of HER2-MCF10A cells using an siRNA molecule targeting leptin. Taken together, the results indicate a molecular link between HER2 and leptin, providing supporting evidence that leptin represents a target for breast cancer therapy. [BMB Reports 2012; 45(12): 719-723] PMID:23261058

  2. Immunogenicity and immunomodulatory properties of hepatocyte-like cells derived from human amniotic epithelial cells.

    PubMed

    Tee, Jing Yang; Vaghjiani, Vijesh; Liu, Yu Han; Murthi, Padma; Chan, James; Manuelpillai, Ursula

    2013-01-01

    Hepatocyte transplantation is being trialled as an alternative to whole organ transplant for patients with acute liver failure and liver specific metabolic diseases. Due to the scarcity of human hepatocytes, hepatocyte-like cells (HLC) generated from stem cells may become a viable alternative to hepatocyte transplantation. Human amniotic epithelial cells (hAEC) from the placenta have stem cell-like properties and can be differentiated into HLC. Naïve hAEC have low immunogenicity and exert immunomodulatory effects that may facilitate allogeneic transplantation. However, whether the immunogenicity and immunomodulatory properties alter with differentiation into HLC are unknown. We further characterized HLC generated from hAEC, examined changes in human leucocyte antigens (HLA) and co-stimulatory molecules and effects exerted by the HLC on human peripheral blood mononuclear cells (PBMC). HLC derived from hAEC expressed proteins found in hepatocytes, had CYP3A4 drug metabolizing enzyme activity and secreted urea. IFN-γ treatment increased HLA Class IA, Class II and co-stimulatory molecule CD40 expression in the HLC. IFN-γ treated HLC stimulated proliferation of PBMC in one-way mixed lymphocyte reactions and were more immunogenic than undifferentiated hAEC. However, the HLC showed immunomodulatory properties and inhibited mitogen induced PBMC proliferation in vitro. PBMC proliferation may have been inhibited by IL-6, TGF-β1, PGE2 and HLA-G secreted by the HLC. The retention of immunomodulatory properties may enable HLC grafts to survive for longer periods despite the immunogenicity of the HLC.

  3. Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells.

    PubMed

    Poma, Anna; Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

    2017-01-01

    Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn't differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication.

  4. Inhibition of interleukin-17-stimulated interleukin-6 and -8 production by cranberry components in human gingival fibroblasts and epithelial cells.

    PubMed

    Tipton, D A; Cho, S; Zacharia, N; Dabbous, M K

    2013-10-01

    Gingival epithelial cells and fibroblasts participate in periodontal inflammation and destruction, producing interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8. IL-17, a product of T-helper 17 cells, may play a role in periodontitis by stimulating cytokine production by gingival cells. The cranberry (Vaccinium macrocarpon) is rich in polyphenols, particularly proanthocyanidins, which have antioxidant and other beneficial properties. Cranberry components inhibit pro-inflammatory activities of lipopolysaccharide-stimulated human macrophages, gingival fibroblasts, and epithelial cells, but little is known of its effects on IL-17-stimulated cytokine production. The objectives were to determine the effects of IL-17 ± cranberry components on IL-6 and IL-8 production by human gingival epithelial cells and fibroblasts. Cranberry high molecular weight non-dialyzable material (NDM), which is rich in proanthocyanidins, was derived from cranberry juice. Human gingival epithelial cells and normal human gingival fibroblasts were incubated with NDM (5-50 μg/mL), IL-17 (0.5-100 ng/mL), or NDM + IL-17 in serum-free medium for 6 d. IL-6 and IL-8 in culture supernatants were measured by ELISA. Membrane damage and viability were assessed by lactate dehydrogenase activity released into cell supernatants and activity of a mitochondrial enzyme, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. In both cell lines, IL-17 (≥ ~5-10 ng/mL) significantly stimulated production of IL-6 (p < 0.005) and IL-8 (p < 0.03). Non-toxic levels of NDM inhibited constitutive IL-6 and IL-8 production by epithelial cells (p ≤ 0.01) and fibroblasts (p ≤ 0.03) as well as IL-17-stimulated cytokine production by epithelial cells [IL-6 (maximum ~80% inhibition; p ≤ 0.0001); IL-8 (maximum ~70% inhibition; p ≤ 0.03)] and fibroblasts [IL-6 (maximum ~90% inhibition; p ≤ 0.0001); IL

  5. Expression of nestin, mesothelin and epithelial membrane antigen (EMA) in developing and adult human meninges and meningiomas.

    PubMed

    Petricevic, Josko; Forempoher, Gea; Ostojic, Ljerka; Mardesic-Brakus, Snjezana; Andjelinovic, Simun; Vukojevic, Katarina; Saraga-Babic, Mirna

    2011-11-01

    The spatial and temporal pattern of appearance of nestin, epithelial membrane antigen (EMA) and mesothelin proteins was immunohistochemically determined in the cells of normal developing and adult human meninges and meningiomas. Human meninges developed as two mesenchymal condensations in the head region. The simple squamous epithelium on the surface of leptomeninges developed during mesenchymal to epithelial transformation. Nestin appeared for the first time in week 7, EMA in week 8, while mesothelin appeared in week 22 of development. In the late fetal period and after birth, nestin expression decreased, whereas expression of EMA and mesothelin increased. EMA appeared in all surface epithelial cells and nodules, while mesothelin was found only in some of them. In adult meninges, all three proteins were predominantly localized in the surface epithelium and meningeal nodules. In meningothelial meningiomas (WHO grade I), EMA was detected in all tumor cells except in the endothelial cells, mesothelin characterized nests of tumor cells, while nestin was found predominantly in the walls of blood vessels. The distribution pattern of those proteins in normal meningeal and tumor cells indicates that nestin might characterize immature cells, while EMA and mesothelin appeared in maturing epithelial cells. Neoplastic transformation of these specific cell lineages contributes to the cell population in meningiomas. Copyright © 2010 Elsevier GmbH. All rights reserved.

  6. Human Urinary Epithelial Cells as a Source of Engraftable Hepatocyte-Like Cells Using Stem Cell Technology.

    PubMed

    Sauer, Vanessa; Tchaikovskaya, Tatyana; Wang, Xia; Li, Yanfeng; Zhang, Wei; Tar, Krisztina; Polgar, Zsuzsanna; Ding, Jianqiang; Guha, Chandan; Fox, Ira J; Roy-Chowdhury, Namita; Roy-Chowdhury, Jayanta

    2016-12-13

    Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps), the method for generating such cells from renal tubular epithelial cells shed in human urine and transplanting them into animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleled that of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant human SERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps that could be potentially useful for disease modeling, pharmacological development, and regenerative medicine.

  7. Paramyxovirus Infection Mimics In Vivo Cellular Dynamics in Three-Demensional Human Bronchio-Epithelial Tissue-Like Assemblies

    NASA Technical Reports Server (NTRS)

    Deatly, Anne M.; Lin, Yen-Huei; McCarthy, Maureen; Chen, Wei; Miller, Lynn Z.; Quiroz, Jorge; Nowak, Becky M.; Lerch, Robert A.; Udem, Stephen A.; Goodwin, Thomas J.

    2012-01-01

    Respiratory syncytial virus and parainfluenza virus cause severe respiratory disease, especially in infants, children and the elderly. An in vitro model that accurately mimics infection of the human respiratory epithelium (HRE) would facilitate vaccine development greatly. Monolayer cultures traditionally used to study these viruses do not accurately and precisely differentiate the replication efficiencies of wild type and attenuated viruses. Therefore, we engineered novel three-dimensional (3D) tissue-like assemblies (TLAs) of human broncho-epithelial (HBE) cells to produce a more physiologically relevant in vitro model of the HRE. TLAs resemble HRE structurally and by expression of differentiated epithelial cell markers. Most significantly, wild type viruses exhibited a clear growth advantage over attenuated strains in TLAs unlike monolayer cultures. In addition, the TLAs responded to virus infection by secreting pro-inflammatory mediators similar to the respiratory epithelia of infected children. These characteristics make the TLA model a valuable platform technology to develop and evaluate live, attenuated respiratory virus vaccine candidates for human use. Respiratory virus diseases, the most frequent and least preventable of all infectious diseases, range in severity from the common cold to severe bronchiolitis and pneumonia . Two paramyxoviruses, respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3), are responsible for a majority of the most severe respiratory diseases of infants and young children. RSV causes 70% of all bronchiolitis cases and is a major cause of morbidity and mortality worldwide, especially in infants. PIV3 causes 10-15% of bronchiolitis and pneumonia during infancy, second only to RSV, and 40% of croup in infants To date, licensed vaccines are not available to prevent these respiratory diseases. At present, traditional monkey kidney (Vero and LLC-MK2) and human (HEp-2) tissue culture cells and small animal models (mouse

  8. An investigation into the concurrent collection of human scent and epithelial skin cells using a non-contact sampling device.

    PubMed

    Caraballo, Norma Iris; Mendel, Julian; Holness, Howard; La Salvia, Joel; Moroose, Tina; Eckenrode, Brian; Stockham, Rex; Furton, Kenneth; Mills, DeEtta

    2016-09-01

    In criminal investigations, the collection of human scent often employs a non-contact, dynamic airflow device, known as the Scent Transfer Unit 100 (STU-100), to transfer volatile organic compounds (VOCs) from an object/person onto a collection material that is subsequently presented to human scent discriminating canines. Human scent is theorized to be linked to epithelial skin cells that are shed at a relatively constant rate allowing both scent and cellular material to be deposited into the environment and/or onto objects. Simultaneous collection of cellular material, with adequate levels of nuclear deoxyribonucleic acid (nDNA), and human scent using a non-invasive methodology would facilitate criminal investigations. This study evaluated the STU-100 for the concurrent collection of human scent and epithelial skin cells from a porous (paper) and non-porous (stainless steel bar) object that was held for a specified period of time in the dominant hand of twenty subjects (10 females and 10 males). Human scent analysis was performed using headspace static solid-phase microextraction with gas chromatography-mass spectrometry (HS-SPME/GC-MS). A polycarbonate filter was used to trap epithelial skin cells which, upon extraction, were subsequently analyzed, inter-laboratory, using the quantitative polymerase chain reaction (qPCR). The STU-100 proved to be inadequate for collecting the minimum number of epithelial skin cells required to obtain nuclear DNA concentrations above the limit of detection for the qPCR kit. With regard to its use for human scent collection, a reduction in the number and mass of compounds was observed when compared to samples that were directly collected. However, when the indirect collection of human scent from the two different objects was compared, a greater number and mass of compounds was observed from the non-porous object than from the porous object. This outcome suggests that the matrix composition of the scent source could affect the

  9. Mechanisms underlying modulation of monocarboxylate transporter 1 (MCT1) by somatostatin in human intestinal epithelial cells.

    PubMed

    Saksena, Seema; Theegala, Saritha; Bansal, Nikhil; Gill, Ravinder K; Tyagi, Sangeeta; Alrefai, Waddah A; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

    2009-11-01

    Somatostatin (SST), an important neuropeptide of the gastrointestinal tract has been shown to stimulate sodium chloride absorption and inhibit chloride secretion in the intestine. However, the effects of SST on luminal butyrate absorption in the human intestine have not been investigated. Earlier studies from our group and others have shown that monocarboxylate transporter (MCT1) plays an important role in the transport of butyrate in the human intestine. The present studies were undertaken to examine the effects of SST on butyrate uptake utilizing postconfluent human intestinal epithelial Caco2 cells. Apical SST treatment of Caco-2 cells for 30-60 min significantly increased butyrate uptake in a dose-dependent manner with maximal increase at 50 nM ( approximately 60%, P < 0.05). SST receptor 2 agonist, seglitide, mimicked the effects of SST on butyrate uptake. SST-mediated stimulation of butyrate uptake involved the p38 MAP kinase-dependent pathway. Kinetic studies demonstrated that SST increased the maximal velocity (V(max)) of the transporter by approximately twofold without any change in apparent Michaelis-Menten constant (K(m)). The higher butyrate uptake in response to SST was associated with an increase in the apical membrane levels of MCT1 protein parallel to a decrease in the intracellular MCT1 pool. MCT1 has been shown to interact specifically with CD147 glycoprotein/chaperone to facilitate proper expression and function of MCT1 at the cell surface. SST significantly enhanced the membrane levels of CD147 as well as its association with MCT1. This association was completely abolished by the specific p38 MAP kinase inhibitor, SB203580. Our findings demonstrate that increased MCT1 association with CD147 at the apical membrane in response to SST is p38 MAP kinase dependent and underlies the stimulatory effects of SST on butyrate uptake.

  10. Sodium metavanadate exhibits carcinogenic tendencies in vitro in immortalized human bronchial epithelial cells.

    PubMed

    Passantino, Lisa; Muñoz, Alexandra B; Costa, Max

    2013-10-01

    Pentavalent vanadium compounds induce intracellular changes in vitro that are consistent with those of other carcinogenic substances. While there is no clear evidence that vanadium compounds cause cancer in humans, vanadium pentoxide causes lung cancer in rodents after long-term inhalation exposures and in turn IARC has categorized it as a group 2B possible human carcinogen. The goal of this study was to investigate the carcinogenicity of NaVO3 in the human immortalized bronchial epithelial cell line, Beas-2B. Cells were treated with 10 μM NaVO3 for 5 weeks, with or without recovery time, followed by gene expression microarray analysis. In a separate experiment, cells were exposed to 1-10 μM NaVO3 for 4 weeks and then grown in soft agar to test for anchorage-independent growth. A dose-dependent increase in the number of colonies was observed. In scratch tests, NaVO3-transformed clones could repair a wound faster than controls. In a gene expression microarray analysis of soft agar clones there were 2010 differentially expressed genes (DEG) (adjusted p-value ≤ 0.05) in NaVO3-transformed clones relative to control clones. DEG from this experiment were compared with the DEG of 5 week NaVO3 exposure with or without recovery, all with adjusted p-values < 0.05, and 469 genes were altered in the same direction for transformed clones, 5 week NaVO3-treated cells, and the recovered cells. The data from this study imply that chronic exposure to NaVO3 causes changes that are consistent with cellular transformation including anchorage-independent growth, enhanced migration ability, and gene expression changes that were likely epigenetically inherited.

  11. Cytoprotective effect of lacritin on human corneal epithelial cells exposed to benzalkonium chloride in vitro

    PubMed Central

    Feng, Mary M.; Baryla, Julia; Liu, Hong; Laurie, Gordon W.; McKown, Robert L.; Ashki, Negin; Bhayana, Dinesh

    2015-01-01

    Purpose Benzalkonium chloride (BAK) is the most commonly found preservative in eye drops, and has been shown to cause ocular surface inflammation and toxicity. Lacritin is a human tear glycoprotein secreted from the lacrimal glands that has been found to be cytoprotective. This study was designed to determine if the presence of lacritin confers protection to a cultured human corneal epithelial (HCE) cell line, CRL-11515, and primary HCE cells after exposure to the ocular preservative agent BAK. Materials and Methods Recombinant human lacritin was cloned into intein fusion vectors, expressed in E. coli, and purified on chitin beads and DEAE Sepharose. Metabolic curves were established using the MTT assay after exposure of subconfluent CRL-11515 cells to BAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II) provided a measure of autophagy in CRL-11515 cells exposed to lacritin and/or BAK. Results BAK reduced CRL-11515 cellular metabolic activity in a time and dose dependent manner. BAK-induced cellular stress was evident by elevated autophagy that increased with rising concentrations of BAK compared to control (P < 0.05). Lacritin increased HCE cell proliferation at an optimal dose of 1 nM. Preconditioning HCE cells with 1 nM lacritin for 24 hours prior to BAK exposure significantly dampened levels of LC3-II (P < 0.05) and promoted a significant increase in cellular metabolic activity (P < 0.01) compared to BAK alone. Conclusions These results suggest lacritin protects cultured HCE cells stressed with BAK. Lacritin may have the potential to be used as a topical adjunctive therapy in eyes chronically exposed to BAK. PMID:24401093

  12. Cytoprotective effect of lacritin on human corneal epithelial cells exposed to benzalkonium chloride in vitro.

    PubMed

    Feng, Mary M; Baryla, Julia; Liu, Hong; Laurie, Gordon W; McKown, Robert L; Ashki, Negin; Bhayana, Dinesh; Hutnik, Cindy M L

    2014-06-01

    Benzalkonium chloride (BAK) is the most commonly found preservative in eye drops, and has been shown to cause ocular surface inflammation and toxicity. Lacritin is a human tear glycoprotein secreted from the lacrimal glands that has been found to be cytoprotective. This study was designed to determine if the presence of lacritin confers protection to a cultured human corneal epithelial (HCE) cell line, CRL-11515, and primary HCE cells after exposure to the ocular preservative agent BAK. Recombinant human lacritin was cloned into intein fusion vectors, expressed in E. coli, and purified on chitin beads and DEAE Sepharose. Metabolic curves were established using the MTT assay after exposure of sub-confluent CRL-11515 cells to BAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II) provided a measure of autophagy in CRL-11515 cells exposed to lacritin and/or BAK. BAK reduced CRL-11515 cellular metabolic activity in a time- and dose-dependent manner. BAK-induced cellular stress was evident by elevated autophagy that increased with rising concentrations of BAK compared to control (p < 0.05). Lacritin increased HCE cell proliferation at an optimal dose of 1 nM. Preconditioning HCE cells with 1 nM lacritin for 24 h prior to BAK exposure significantly dampened levels of LC3-II (p < 0.05) and promoted a significant increase in cellular metabolic activity (p < 0.01) compared to BAK alone. These results suggest lacritin protects cultured HCE cells stressed with BAK. Lacritin may have the potential to be used as a topical adjunctive therapy in eyes chronically exposed to BAK.

  13. KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts.

    PubMed

    Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C; Warwar, Ronald; Brown, Thomas L; Lauf, Peter K

    2006-11-01

    We recently reported potassium-chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium-chloride cotransport (KCC) isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT-PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100kDa band in HLE-B3 cells. In the human lens tissues, a 115kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.

  14. Differential Response of Human Nasal and Bronchial Epithelial Cells upon Exposure to Size-fractionated Dairy Dust

    PubMed Central

    Hawley, Brie; Schaeffer, Joshua; Poole, Jill A.; Dooley, Gregory P.; Reynolds, Stephen; Volckens, John

    2015-01-01

    Exposure to organic dusts is associated with increased respiratory morbidity and mortality in agricultural workers. Organic dusts in dairy farm environments are complex, polydisperse mixtures of toxic and immunogenic compounds. Previous toxicological studies focused primarily on exposures to the respirable size fraction, however, organic dusts in dairy farm environments are known to contain larger particles. Given the size distribution of dusts from dairy farm environments, the nasal and bronchial epithelia represent targets of agricultural dust exposures. In this study, well-differentiated normal human bronchial epithelial cells and human nasal epithelial cells were exposed to two different size fractions (PM10 and PM>10) of dairy parlor dust using a novel aerosol-to-cell exposure system. Levels of pro-inflammatory transcripts (IL-8, IL-6, and TNF-α) were measured two hr after exposure. Lactate dehydrogenase (LDH) release was also measured as an indicator of cytotoxicity. Cell exposure to dust was measured in each size fraction as a function of mass, endotoxin, and muramic acid levels. To our knowledge, this is the first study to evaluate the effects of distinct size fractions of agricultural dust on human airway epithelial cells. Our results suggest that both PM10 and PM>10 size fractions elicit a pro-inflammatory response in airway epithelial cells and that the entire inhalable size fraction needs to be considered when assessing potential risks from exposure to agricultural dusts. Further, data suggest that human bronchial cells respond differently to these dusts than human nasal cells and, therefore, the two cell types need to be considered separately in airway cell models of agricultural dust toxicity. PMID:25965193

  15. Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells.

    PubMed

    Li, Yanwei; Liu, Haifeng; Zeng, Wei; Wei, Jing

    2017-01-01

    An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.

  16. Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells

    PubMed Central

    Li, Yanwei; Liu, Haifeng; Zeng, Wei; Wei, Jing

    2017-01-01

    An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC. PMID:28346481

  17. Matrix regeneration agents improve wound healing in non-stressed human corneal epithelial cells.

    PubMed

    Robciuc, A; Arvola, R P J; Jauhiainen, M; Holopainen, J M

    2018-04-01

    PurposeMatrix regenerating agents (RGTAs) emerged as promising in vivo wound-healing agents. These agents could prove beneficial for the treatment of dry eye disease-associated corneal micro-erosions; therefore, we aimed to evaluate the wound healing efficacy of regenerative agents (RGTAs or serum) in an in vitro model of hyperosmolarity (HO) stressed and non-stressed human corneal epithelial cells.Patients and methodsThe migration and proliferation induced by the regenerative agents was evaluated using an in vitro scratch wound assay and brome-deoxy-uridine incorporation. The inflammatory profile and effects of osmoregulators were also investigated. The two-tailed paired t-test calculated the statistical significance, with P-value<0.05 considered significant.ResultsThe most efficient inducer of re-epithelization was 2% serum, followed closely by 2% RGTA with an average improvement in cell migration of 1.8- and 1.4-fold, respectively, when compared with the non-treated control. Hyperosmolar stress significantly reduced the restorative effects of both serum and RGTAs; these effects were, however, neutralized by the osmoregulator betaine.ConclusionThese findings suggest that RGTAs could provide efficient treatment for dry-eye associated corneal micro-lesions if ocular surface HO is neutralized.

  18. Cultured Human Retinal Pigment Epithelial (hRPE) Sheets: A Search for Suitable Storage Conditions.

    PubMed

    Khan, Ayyad Z; Utheim, Tor P; Reppe, Sjur; Sandvik, Leiv; Lyberg, Torstein; Roald, Borghild B-H; Ibrahim, Ibrahim B; Eidet, Jon R

    2018-04-01

    The advancement of human retinal pigment epithelial cell (hRPE) replacement therapy is partly dependent on optimization of cell culture, cell preservation, and storage medium. This study was undertaken to search for a suitable storage temperature and storage medium for hRPE. hRPE monolayer sheets were cultured under standard conditions at 37°C and then randomized for storage at six temperatures (4, 16, 20, 24, 28, and 37°C) for 7 days. After revealing a suitable storage temperature, hRPE sheets were subsequently stored with and without the silk protein sericin added to the storage medium. Live/dead assay, light microscopy, pH, and phenotypic expression of various proteins were used to assess cell cultures stored at different temperatures. After 7 days of storage, hRPE morphology was best preserved at 4°C. Addition of sericin to the storage medium maintained the characteristic morphology of the preserved cells, and improved pigmentation and levels of pigmentation-related proteins in the cultured hRPE sheets following a 7-day storage period at 4°C.

  19. [Effect of ultraviolet radiation on ALDH1 expression in human lens epithelial cells].

    PubMed

    Shi, Jingming; Jia, Songbai; Chen, Xuan; Tang, Luosheng

    2012-06-01

    To determine the apoptosis-inducing effect of ultraviolet light (UV) on human lens epithelial cell (HLEC) and to explore the involvement of changes in ALDH1 folowing UV radiation. HLEC was exposed to the same UV light source and was subsequently divided into 6 groups according to UV radiation time of 0 (control group), 5, 10, 15, and 30 min. Apoptosis was detected by AO/EB staining. Changes of ALDH1 in HLEC were detected by immunohistochemical staining and Western blot. The intensity of immunohistochemical staining and the rate of positive cells decreased with increase of UV time (P<0.05). The rate of positive ALDH1 cells was negatively correlated with the rate of apoptosis (r= -0.92, P<0.05). Western blot showed the integrated absorbance values significantly decreased with the increase of UV time (P<0.05). ALDH1 in HLEC decreases with an increase of UV exposure, which may be related to UV induced apoptosis of HLEC.

  20. [Effect of flavin adenine dinucleotide on ultraviolet B induced damage in cultured human corneal epithelial cells].

    PubMed

    Sakamoto, Asuka; Nakamura, Masatsugu

    2012-01-01

    This study evaluated the effects of flavin adenine dinucleotide (FAD) on ultraviolet B (UV-B)-induced damage in cultured human corneal epithelial (HCE-T) cells. The cultured HCE-T cells were treated with 0.003125-0.05% FAD before exposure to 80 mJ/cm2 UV-B. Cell viability was measured 24 h after UV-B irradiation using the MTS assay. Reactive oxygen species (ROS) were detected 30 min after UV-B irradiation using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester. Apoptosis was evaluated 4 h after UV-B irradiation in the caspase-3/7 activity assay. UV-B irradiation reduced cell viability and stimulated ROS production and caspase-3/7 activity in HCE-T cells. Pretreatment of UV-B irradiated HCE-T cells with FAD significantly attenuated cell viability reduction and inhibited the stimulation of both ROS production and caspase-3/7 activity due to UV-B exposure compared with those with vehicle (0% FAD). These results clarified that FAD inhibits ROS-mediated apoptosis by UV-B irradiation in HCE-T cells and suggest that FAD may be effective as a radical scavenger in UV-B-induced corneal damage.

  1. Arsenic Alters ATP-Dependent Ca2+ Signaling in Human Airway Epithelial Cell Wound Response

    PubMed Central

    Sherwood, Cara L.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2011-01-01

    Arsenic is a natural metalloid toxicant that is associated with occupational inhalation injury and contaminates drinking water worldwide. Both inhalation of arsenic and consumption of arsenic-tainted water are correlated with malignant and nonmalignant lung diseases. Despite strong links between arsenic and respiratory illness, underlying cell responses to arsenic remain unclear. We hypothesized that arsenic may elicit some of its detrimental effects on the airway through limitation of innate immune function and, specifically, through alteration of paracrine ATP (purinergic) Ca2+ signaling in the airway epithelium. We examined the effects of acute (24 h) exposure with environmentally relevant levels of arsenic (i.e., < 4μM as Na-arsenite) on wound-induced Ca2+ signaling pathways in human bronchial epithelial cell line (16HBE14o-). We found that arsenic reduces purinergic Ca2+ signaling in a dose-dependent manner and results in a reshaping of the Ca2+ signaling response to localized wounds. We next examined arsenic effects on two purinergic receptor types: the metabotropic P2Y and ionotropic P2X receptors. Arsenic inhibited both P2Y- and P2X-mediated Ca2+ signaling responses to ATP. Both inhaled and ingested arsenic can rapidly reach the airway epithelium where purinergic signaling is essential in innate immune functions (e.g., ciliary beat, salt and water transport, bactericide production, and wound repair). Arsenic-induced compromise of such airway defense mechanisms may be an underlying contributor to chronic lung disease. PMID:21357385

  2. Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells

    PubMed Central

    Mandlik, Anjali; Swierczynski, Arlene; Das, Asis; Ton-That, Hung

    2010-01-01

    Summary Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection. PMID:17376076

  3. Combined Effects of Nonylphenol and Bisphenol A on the Human Prostate Epithelial Cell Line RWPE-1

    PubMed Central

    Gan, Weidong; Zhou, Ming; Xiang, Zou; Han, Xiaodong; Li, Dongmei

    2015-01-01

    The xenoestrogens nonylphenol (NP) and bisphenol A (BPA) are regarded as endocrine disrupting chemicals (EDCs) which have widespread occurrence in our daily life. In the present study, the purpose was to analyze the combined effects of NP and BPA on the human prostate epithelial cell line RWPE-1 using two mathematical models based on the Loewe additivity (LA) theory and the Bliss independence (BI) theory. RWPE-1 cells were treated with NP (0.01–100 µM) and BPA (1–5000 µM) in either a single or a combined format. A cell viability assay and lactate dehydrogenase (LDH) leakage rate assay were employed as endpoints. As predicted by the two models and based on the cell viability assay, significant synergism between NP and BPA were observed. However, based on the LDH assay, the trends were reversed. Given that environmental contaminants are frequently encountered simultaneously, these data indicated that there were potential interactions between NP and BPA, and the combined effects of the chemical mixture might be stronger than the additive values of individual chemicals combined, which should be taken into consideration for the risk assessment of EDCs. PMID:25874684

  4. Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

    PubMed Central

    Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

    2015-01-01

    The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting. PMID:26068810

  5. Human papillomavirus (HPV)-18 E6 oncoprotein interferes with the epithelial cell polarity Par3 protein.

    PubMed

    Facciuto, Florencia; Bugnon Valdano, Marina; Marziali, Federico; Massimi, Paola; Banks, Lawrence; Cavatorta, Ana Laura; Gardiol, Daniela

    2014-05-01

    High-risk human papillomavirus (HPV) infection is the principal risk factor for the development of cervical cancer. The HPV E6 oncoprotein has the ability to target and interfere with several PSD-95/DLG/ZO-1 (PDZ) domain-containing proteins that are involved in the control of cell polarity. This function can be significant for E6 oncogenic activity because a deficiency in cell polarisation is a marker of tumour progression. The establishment and control of polarity in epithelial cells depend on the correct asymmetrical distribution of proteins and lipids at the cell borders and on specialised cell junctions. In this report, we have investigated the effects of HPV E6 protein on the polarity machinery, with a focus on the PDZ partitioning defective 3 (Par3) protein, which is a key component of tight junctions (TJ) and the polarity network. We demonstrate that E6 is able to bind and induce the mislocalisation of Par3 protein in a PDZ-dependent manner without significant reduction in Par3 protein levels. In addition, the high-risk HPV-18 E6 protein promotes a delay in TJ formation when analysed by calcium switch assays. Taken together, the data presented in this study contribute to our understanding of the molecular mechanism by which HPVs induce the loss of cell polarity, with potential implications for the development and progression of HPV-associated tumours. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. TRIM24 links glucose metabolism with transformation of human mammary epithelial cells.

    PubMed

    Pathiraja, T N; Thakkar, K N; Jiang, S; Stratton, S; Liu, Z; Gagea, M; Shi, X; Shah, P K; Phan, L; Lee, M-H; Andersen, J; Stampfer, M; Barton, M C

    2015-05-28

    Tripartite motif 24 protein (TRIM24) is a plant homeodomain/bromodomain histone reader, recently associated with poor overall survival of breast-cancer patients. At a molecular level, TRIM24 is a negative regulator of p53 levels and a co-activator of estrogen receptor. However, the role of TRIM24 in breast tumorigenesis remains largely unknown. We used an isogenic human mammary epithelial cell (HMEC) culture model, derived from reduction mammoplasty tissue, and found that ectopic expression of TRIM24 in immortalized HMECs (TRIM24 iHMECs) greatly increased cellular proliferation and induced malignant transformation. Subcutaneous injection of TRIM24 iHMECs in nude mice led to growth of intermediate to high-grade tumors in 60-70% of mice. Molecular analysis of TRIM24 iHMECs revealed a glycolytic and tricarboxylic acid cycle gene signature, alongside increased glucose uptake and activated aerobic glycolysis. Collectively, these results identify a role for TRIM24 in breast tumorigenesis through reprogramming of glucose metabolism in HMECs, further supporting TRIM24 as a viable therapeutic target in breast cancer.

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  8. Establishment and transformation of telomerase-immortalized human small airway epithelial cells by heavy ions

    NASA Astrophysics Data System (ADS)

    Zhao, Y. L.; Piao, C. Q.; Hei, T. K.

    Previous studies from this laboratory have identified a number of causally linked genes including the novel tumor suppressor Betaig-h3 that were differentially expressed in radiation induced tumorigenic BEP2D cells. To extend these studies using a genomically more stable bronchial cell line, we show here that ectopic expression of the catalytic subunit of telomerase (hTERT) in primary human small airway epithelial (SAE) cells resulted in the generation of several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal. Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings. The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice. These cells show no alteration in the p53 gene but a decrease in p16 expression. Exponentially growing SAEh cells were exposed to graded doses of 1 GeV/nucleon of 56Fe ions accelerated at the Brookhaven National Laboratory. Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation. Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium. These findings indicate that hTERT-immortalized cells, being diploid and chromosomal stable, should be a useful model in assessing mechanism of radiation carcinogenesis.

  9. Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

    PubMed Central

    Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X.

    2008-01-01

    Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-κB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-κB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1) and antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and contributes to innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. PMID:18191935

  10. Protective effect of carbachol postconditioning on hypoxia/reoxygenation-induced injury in human gastric epithelial cells.

    PubMed

    Han, Hongxia; Yang, Jun; Fei, Sujuan; Liu, Zhangbo; Zhu, Shengping; Dong, Qiuju; Gao, Zhifeng; Wang, Shihui; Zhang, Jianfu

    2016-01-01

    We investigated the protective effects of carbachol postconditioning (CAR-P) on acute gastric mucosal injury induced by hypoxia/reoxygenation (H/R) and its possible mechanisms. Cell viability was detected by methyl thiazolyl tetrazolium (MTT). The apoptotic cells were examined by Hoechst 33258 staining. Flow cytometric analysis, lactate dehydrogenate (LDH) release assay, immunocytochemistry, and western blotting were used to investigate the effects of CAR-P on acute gastric mucosal injury induced by H/R. The model of H/R was established by hypoxia induction(94% N2+1% O2+5% CO2 for 2 h) and reoxygenation (normoxic condition for 4 h, 8 h and 16 h). Our study observed the protective effect of carbachol postconditioning on H/R-induced injury in human gastric epithelial cell lines (hGES-1) cells, which is achieved by direct activation of vanilloid receptor subtype 1 (VR1) and production of calcitonin gene-related peptide (CGRP), and in the inhibition of cell apoptosis. In the study, we demonstrate that CAR-P has protective effects on the H/R-induced injury in hGES-1 cells, and these effects are associated with cholinergic muscarinic receptors (CMR), VR1, and extracellular signal-regulated kinase (ERK) signaling pathway. Our findings might provide a new and improved understanding of CAR-P function and an effective treatment strategy for acute gastric mucosal injury induced by H/R. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Lesion complexity drives age related cancer susceptibility in human mammary epithelial cells

    DOE PAGES

    Sridharan, Deepa M.; Enerio, Shiena; Stampfer, Martha M.; ...

    2017-02-28

    Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by one's age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations andmore » changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population.« less

  12. Titanium dioxide nanoparticles activate IL8-related inflammatory pathways in human colonic epithelial Caco-2 cells

    NASA Astrophysics Data System (ADS)

    Krüger, Kristin; Cossais, François; Neve, Horst; Klempt, Martin

    2014-05-01

    Nanosized titanium dioxide (TiO2) particles are widely used as food additive or coating material in products of the food and pharmaceutical industry. Studies on various cell lines have shown that TiO2 nanoparticles (NPs) induced the inflammatory response and cytotoxicity. However, the influences of TiO2 NPs' exposure on inflammatory pathways in intestinal epithelial cells and their differentiation have not been investigated so far. This study demonstrates that TiO2 NPs with particle sizes ranging between 5 and 10 nm do not affect enterocyte differentiation but cause an activation of inflammatory pathways in the human colon adenocarcinoma cell line Caco-2. 5 and 10 nm NPs' exposures transiently induce the expression of ICAM1, CCL20, COX2 and IL8, as determined by quantitative PCR, whereas larger particles (490 nm) do not. Further, using nuclear factor (NF)-κB reporter gene assays, we show that NP-induced IL8 mRNA expression occurs, in part, through activation of NF-κB and p38 mitogen-activated protein kinase pathways.

  13. Paracrine influence of human perivascular cells on the proliferation of adenocarcinoma alveolar epithelial cells.

    PubMed

    Kim, Eunbi; Na, Sunghun; An, Borim; Yang, Se-Ran; Kim, Woo Jin; Ha, Kwon-Soo; Han, Eun-Taek; Park, Won Sun; Lee, Chang-Min; Lee, Ji Yoon; Lee, Seung-Joon; Hong, Seok-Ho

    2017-03-01

    Understanding the crosstalk mechanisms between perivascular cells (PVCs) and cancer cells might be beneficial in preventing cancer development and metastasis. In this study, we investigated the paracrine influence of PVCs derived from human umbilical cords on the proliferation of lung adenocarcinoma epithelial cells (A549) and erythroleukemia cells (TF-1α and K562) in vitro using Transwell® co-culture systems. PVCs promoted the proliferation of A549 cells without inducing morphological changes, but had no effect on the proliferation of TF-1α and K562 cells. To identify the factors secreted from PVCs, conditioned media harvested from PVC cultures were analyzed by antibody arrays. We identified a set of cytokines, including persephin (PSPN), a neurotrophic factor, and a key regulator of oral squamous cell carcinoma progression. Supplementation with PSPN significantly increased the proliferation of A549 cells. These results suggested that PVCs produced a differential effect on the proliferation of cancer cells in a cell-type dependent manner. Further, secretome analyses of PVCs and the elucidation of the molecular mechanisms could facilitate the discovery of therapeutic target(s) for lung cancer.

  14. BAG3 regulates epithelial-mesenchymal transition and angiogenesis in human hepatocellular carcinoma.

    PubMed

    Xiao, Heng; Cheng, Shaobing; Tong, Rongliang; Lv, Zheng; Ding, Chaofeng; Du, Chengli; Xie, Haiyang; Zhou, Lin; Wu, Jian; Zheng, Shusen

    2014-03-01

    Bcl2-associated athanogene 3 (BAG3) protein is a co-chaperone of heat-shock protein (Hsp) 70 and may regulate major physiological and pathophysiological processes. However, few reports have examined the role of BAG3 in human hepatocellular carcinoma (HCC). In this study, we show that BAG3 regulates epithelial-mesenchymal transition (EMT) and angiogenesis in HCC. BAG3 was overexpressed in HCC tissues and cell lines. BAG3 knockdown resulted in reduction in migration and invasion of HCC cells, which was linked to reversion of EMT by increasing E-cadherin expression and decreasing N-cadherin, vimentin and slug expression, as well as suppressing matrix metalloproteinase 2 (MMP-2) expression. In a xenograft tumorigenicity model, BAG3 knockdown effectively inhibited tumor growth and metastasis through reduction in CD34 and VEGF expression and reversal of the EMT pathway. In conclusion, BAG3 is associated with the invasiveness and angiogenesis in HCC, and the BAG3 gene may be a novel therapeutic approach against HCC.

  15. Mechanistic insights into the impact of Cold Atmospheric Pressure Plasma on human epithelial cell lines

    NASA Astrophysics Data System (ADS)

    Dezest, Marlène; Chavatte, Laurent; Bourdens, Marion; Quinton, Damien; Camus, Mylène; Garrigues, Luc; Descargues, Pascal; Arbault, Stéphane; Burlet-Schiltz, Odile; Casteilla, Louis; Clément, Franck; Planat, Valérie; Bulteau, Anne-Laure

    2017-01-01

    Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPP) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O2, He-N2) and human epithelial cell lines to gain better insight into plasma-cell interaction. We provide evidence that reactive oxygen and nitrogen species (RONS) are inducing cell death by apoptosis and that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target of (He or He-N2) CAPP. However, RONS are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O2 CAPP. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but is also affecting extracellular matrix in keratinocytes. Moreover, we found that malignant cells are more resistant to CAPP treatment than normal cells. Taken together, our findings provide insight into potential mechanisms of CAPP-induced proteasome inactivation and the cellular consequences of these events.

  16. Lesion complexity drives age related cancer susceptibility in human mammary epithelial cells

    SciTech Connect

    Sridharan, Deepa M.; Enerio, Shiena; Stampfer, Martha M.

    Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by one's age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations andmore » changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population.« less

  17. Activation of MAP kinases by hexavalent chromium, manganese and nickel in human lung epithelial cells.

    PubMed

    Tessier, Daniel M; Pascal, Laura E

    2006-12-01

    Epidemiological studies indicate that workers who perform welding operations are at increased risk for bronchitis, siderosis, occupational asthma and lung cancer due to fume exposure. Welding fumes are a complex chemical mixture, and the metal composition is hypothesized to be an etiological factor in respiratory disease due to this exposure. In the present study, human lung epithelial cells in vitro responded to hexavalent chromium, manganese and nickel over a concentration range of 0.2-200 microM with a significant increase in intracellular phosphoprotein (a measure of stress response pathway activation). The mitogen-activated protein kinases ERK1/2, SAPK/JNK and p38 were activated via phosphorylation following 1-h exposures. Hexavalent chromium up-regulated p-38 phosphorylation 23-fold and SAPK/JNK phosphorylation 17-fold, with a comparatively modest 4-fold increase in ERK1/2 phosphorylation. Manganese caused a two- to four-fold increase in SAPK/JNK and ERK 1/2 phosphorylation, with no observed effects on p38 kinase. Nickel caused increased (two-fold) phosphorylation of ERK 1/2 only, and was not cytotoxic over the tested concentration range. The observed effects of welding fume metals on cellular signaling in lung epithelium demonstrate a potentially significant interplay between stress-response signaling (p38 and SAPK/JNK) and anti-apototic signaling (ERK 1/2) that is dependant on the specific metal or combination of metals involved.

  18. The Fate of ZnO Nanoparticles Administered to Human Bronchial Epithelial Cells

    PubMed Central

    Gilbert, Benjamin; Fakra, Sirine C.; Xia, Tian; Pokhrel, Suman; Mädler, Lutz; Nel, André E.

    2014-01-01

    A particular challenge for nanotoxicology is the evaluation of the biological fate and toxicity of nanomaterials that dissolve in aqueous fluids. Zinc oxide nanomaterials are of particular concern because dissolution leads to release of the toxic divalent zinc ion. Although dissolved zinc ions have been implicated in ZnO cytotoxicity, direct identification of the chemical form of zinc taken up by cells exposed to ZnO nanoparticles, and its intracellular fate, has not yet been achieved. We combined high resolution X-ray spectromicroscopy and high elemental sensitivity X-ray microprobe analyses to determine the fate of ZnO and less soluble iron-doped ZnO nanoparticles following exposure to cultures of human bronchial epithelial cells, BEAS-2B. We complemented two-dimensional X-ray imaging methods with atomic force microscopy of cell surfaces to distinguish between nanoparticles that were transported inside the cells from those that adhered to the cell exterior. The data suggest cellular uptake of ZnO nanoparticles is a mechanism of zinc accumulation in cells. Following uptake, ZnO nanoparticles dissolved completely generating intracellular Zn2+ complexed by molecular ligands. These results corroborate a model for ZnO nanoparticle toxicity that is based on nanoparticle uptake followed by intracellular dissolution. PMID:22646753

  19. Calcium ions rescue human lung epithelial cells from the toxicity of zinc oxide nanoparticles.

    PubMed

    Hanagata, Nobutaka; Morita, Hiromi

    2015-01-01

    Contradictory results have been reported for in vitro evaluations of whether zinc oxide nanoparticles (ZnO NPs) are cytotoxic. Though there have been reports of ZnO NPs cytotoxicity due to Zn ions released from the nanoparticles, there have also been reports concluding that Zn ions are not cytotoxic. This inconsistency is mostly attributed to the types of cells used. In this research, we investigated the difference in the level of ZnO NPs cytotoxicity due to culturing conditions. The sensitivity of human lung epithelial cells to ZnO NPs cytotoxicity differed depending on the dispersing medium, physiological state of the cells resulting from their growth stage, and composition of the medium. Further, with regard to the toxicity of ZnO NPs, NPs internalized into cells had a greater cytotoxic effect than Zn ions released from ZnO NPs. Instead of inducing cell death, ZnO NPs internalized into cells slowed the rate of cell proliferation. Furthermore, the cytotoxicity of ZnO NPs depended greatly on the concentration of calcium ions (Ca2+) in the medium. When the concentration of Ca2+ was low, the cytotoxicity of ZnO NPs increased markedly. However, the toxicity of ZnO NPs was mitigated by the addition of CaCl2 to the medium. Global gene expression analysis revealed that Ca2+ -induced upregulation of cell cycle functions could be attributable to the mitigation of ZnO NP toxicity by Ca2+.

  20. Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2.

    PubMed

    Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X

    2008-05-01

    Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappaB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappaB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1), antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules.

  1. Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation

    PubMed Central

    Marconett, Crystal N.; Zhou, Beiyun; Rieger, Megan E.; Selamat, Suhaida A.; Dubourd, Mickael; Fang, Xiaohui; Lynch, Sean K.; Stueve, Theresa Ryan; Siegmund, Kimberly D.; Berman, Benjamin P.

    2013-01-01

    Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation. PMID:23818859

  2. Karyotyping of Chromosomes in Human Bronchial Epithelial Cells Transformed by High Energy Fe Ions

    NASA Technical Reports Server (NTRS)

    Yeshitla, Samrawit; Zhang, Ye; Park, Seongmi; Story, Michael T.; Wilson, Bobby; Wu, Honglu

    2014-01-01

    Lung cancer induced from exposure to space radiation is believed to be one of the most significant health risks for long-term space travels. In a previous study, normal human bronchial epithelial cells (HBECs), immortalized through the expression of Cdk4 and hTERT, were exposed to gamma rays and high energy Fe ions for the selection of transformed clones induced by low- and high-LET radiation. In this research, we analyzed chromosome aberrations in these selected clones for genomic instability using the multi-color fluorescent in situ hybridization (mFISH), as well as the multi-banding in situ hybridization (mBAND) techniques. In most of the clones, we found chromosomal aberrations involving translocations between different chromosomes, with several of the breaks occurred in the q-arm of chromosome 3. We also identified copy number variations between the transformed clones and the parental HBEC cells regardless of the exposure condition. Our results indicated that the chromosomal aberrations in low- and high radiation-induced transformed clones are inadequately different from spontaneous soft agar growth. Further analysis is underway to reveal the genomic instability in more transformed clones

  3. Pirfenidone inhibits migration, differentiation, and proliferation of human retinal pigment epithelial cells in vitro

    PubMed Central

    Wang, Jing; Yang, Yangfan; Xu, Jiangang; Lin, Xianchai; Wu, Kaili

    2013-01-01

    Purpose To investigate the effects of pirfenidone (PFD) on the migration, differentiation, and proliferation of retinal pigment epithelial (RPE) cells and demonstrate whether the drug induces cytotoxicity. Methods Human RPE cells (line D407) were treated with various concentrations of PFD. Cell migration was measured with scratch assay. The protein levels of fibronectin (FN), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), transforming growth factor beta (TGFβS), and Smads were assessed with western blot analyses. Levels of mRNA of TGFβS, FN, and Snail1 were analyzed using reverse transcriptase–polymerase chain reaction. Cell apoptosis was detected with flow cytometry using the Annexin V/PI apoptosis kit, and the percentages of cells labeled in different apoptotic stage were compared. A Trypan Blue assay was used to assess cell viability. Results PFD inhibited RPE cell migration. Western blot analyses showed that PFD inhibited the expression of FN, α-SMA, CTGF, TGFβ1, TGFβ2, Smad2/3, and Smad4. Similarly, PFD also downregulated mRNA levels of Snail1, FN, TGFβ1, and TGFβ2. No significant differences in cell apoptosis or viability were observed between the control and PFD-treated groups. Conclusions PFD inhibited RPE cell migration, differentiation, and proliferation in vitro and caused no significant cytotoxicity. PMID:24415895

  4. Karyotyping of Chromosomes in Human Bronchial Epithelial Cells Transformed by High Energy Fe Ions

    NASA Technical Reports Server (NTRS)

    Yeshitla, Samrawit; Zhang, Ye; Park, Seongmi; Story, Michael D.; Wilson, Bobby; Wu, Honglu

    2015-01-01

    Lung cancer induced from exposures to space radiation is one of the most significant health risks for long-term space travels. Evidences show that low- and high- Linear energy transfer (LET)-induced transformation of normal human bronchial epithelial cells (HBEC) that are immortalized through the expression of Cdk4 and hTERT. The cells were exposed to gamma rays and high-energy Fe ions for the selection of transformed clones. Transformed HBEC are identified and analyzed chromosome aberrations (i.e. genomic instability) using the multi-color fluorescent in situ hybridization (mFISH), as well as the multi-banding in situ hybridization (mBAND) techniques. Our results show chromosomal translocations between different chromosomes and several of the breaks occurred in the q-arm of chromosome 3. We also identified copy number variations between the transformed and the parental HBEC regardless of the exposure conditions. We observed chromosomal aberrations in the lowand high-LET radiation-induced transformed clones and they are imperfectly different from clones obtain in spontaneous soft agar growth.

  5. Organic acids influence iron uptake in the human epithelial cell line Caco-2.

    PubMed

    Salovaara, Susan; Sandberg, Ann-Sofie; Andlid, Thomas

    2002-10-09

    It has previously been suggested that organic acids enhance iron absorption. We have studied the effect of nine organic acids on the absorption of Fe(II) and Fe(III) in the human epithelial cell line Caco-2. The effect obtained was dose-dependent, and the greatest increase (43-fold) was observed for tartaric acid (4 mmol/L) on Fe(III) (10 micromol/L). Tartaric, malic, succinic, and fumaric acids enhanced Fe(II) and Fe(III) uptake. Citric and oxalic acid, on the other hand, inhibited Fe(II) uptake but enhanced Fe(III) uptake. Propionic and acetic acid increased the Fe(II) uptake, but had no effect on Fe(III) uptake. Our results show a correlation between absorption pattern and chemical structure; e.g. hydroxyl groups, in addition to carboxyls, were connected with a positive influence. The results may be important for elucidating factors affecting iron bioavailability in the small intestine and for the development of foods with improved iron bioavailability.

  6. Effects of a human recombinant alkaline phosphatase during impaired mitochondrial function in human renal proximal tubule epithelial cells.

    PubMed

    Peters, Esther; Schirris, Tom; van Asbeck, Alexander H; Gerretsen, Jelle; Eymael, Jennifer; Ashikov, Angel; Adjobo-Hermans, Merel J W; Russel, Frans; Pickkers, Peter; Masereeuw, Rosalinde

    2017-02-05

    Sepsis-associated acute kidney injury is a multifactorial syndrome in which inflammation and renal microcirculatory dysfunction play a profound role. Subsequently, renal tubule mitochondria reprioritize cellular functions to prevent further damage. Here, we investigated the putative protective effects of human recombinant alkaline phosphatase (recAP) during inhibition of mitochondrial respiration in conditionally immortalized human proximal tubule epithelial cells (ciPTEC). Full inhibition of mitochondrial oxygen consumption was obtained after 24h antimycin A treatment, which did not affect cell viability. While recAP did not affect the antimycin A-induced decreased oxygen consumption and increased hypoxia-inducible factor-1α or adrenomedullin gene expression levels, the antimycin A-induced increase of pro-inflammatory cytokines IL-6 and IL-8 was attenuated. Antimycin A tended to induce the release of detrimental purines ATP and ADP, which reached statistical significance when antimycin A was co-incubated with lipopolysaccharide, and were completely converted into cytoprotective adenosine by recAP. As the adenosine A 2A receptor was up-regulated after antimycin A exposure, an adenosine A 2A receptor knockout ciPTEC cell line was generated in which recAP still provided protection. Together, recAP did not affect oxygen consumption but attenuated the inflammatory response during impaired mitochondrial function, an effect suggested to be mediated by dephosphorylating ATP and ADP into adenosine. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Focal epithelial hyperplasia associated with human papillomavirus 13 and common human leukocyte antigen alleles in a Turkish family.

    PubMed

    Akoğlu, Gülşen; Metin, Ahmet; Ceylan, Gülay Güleç; Emre, Selma; Akpolat, Demet; Süngü, Nuran

    2015-02-01

    Focal epithelial hyperplasia (FEH) is a rare and benign papillomatous disease of the oral cavity, which is closely associated with human papillomavirus (HPV) type 13 and 32. Genetic susceptibility to HPV infections are supported by recent studies involving the human leukocyte antigen system (HLA). In this report, we aimed to determine the clinicopathological features of a Turkish family with FEH and to detect the shared HLA DR and DQ types. HPV DNA typing of tissue samples and HLA determination from blood samples of four family members were performed by polymerase chain reaction. Histopathological examination of all patients revealed acanthotic papillomatous epidermis, koilocytes, apoptotic keratinocytes, and mitosoid bodies. HPV13 was detected by polymerase chain reaction. HLA DQA1*0501, HLA DQB1*0302, and HLA DRB1*11 alleles were common in all family members. HLA DRB1*04 was detected in three of them. This report is the first step for the investigation of involvement of HLA types in the pathogenesis of Turkish patients with FEH. © 2014 The International Society of Dermatology.

  8. EMMPRIN Is Secreted by Human Uterine Epithelial Cells in Microvesicles and Stimulates Metalloproteinase Production by Human Uterine Fibroblast Cells

    PubMed Central

    Dayger, C. A.; Mehrotra, P.; Belton, R. J.; Nowak, R. A.

    2012-01-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1β/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C. PMID:22729071

  9. EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells.

    PubMed

    Braundmeier, A G; Dayger, C A; Mehrotra, P; Belton, R J; Nowak, R A

    2012-12-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1β/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C.

  10. Serotonin suppresses β-casein expression via PTP1B activation in human mammary epithelial cells.

    PubMed

    Chiba, Takeshi; Maeda, Tomoji; Sanbe, Atsushi; Kudo, Kenzo

    2016-04-22

    Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of β-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of β-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of β-casein and STAT5 phosphorylation. The β-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 μM db-cAMP for 6 h significantly decreased the protein levels of β-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the β-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of β-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of β-casein expression in MCF-12A cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Hexavalent Chromium Causes the Oxidation of Thioredoxin in Human Bronchial Epithelial Cells

    PubMed Central

    Myers, Judith M.; Antholine, William E.; Myers, Charles R.

    2008-01-01

    Hexavalent chromium [Cr(VI)] species such as chromates are cytotoxic. Inhalational exposure is a primary concern in many Cr-related industries and their immediate environments, and bronchial epithelial cells are directly exposed to inhaled Cr(VI). Chromates are readily taken up by cells and are reduced to reactive Cr species which may also result in the generation of reactive oxygen species (ROS). The thioredoxin (Trx) system has a key role in the maintenance of cellular thiol redox balance and is essential for cell survival. Cells normally maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. Redox western blots were used to assess the redox status of the thioredoxins in normal human bronchial epithelial cells (BEAS-2B) incubated with soluble Na2CrO4 or insoluble ZnCrO4 for different periods of time. Both chromates caused a dose- and time-dependent oxidation of Trx2 and Trx1. Trx2 was more susceptible in that it could all be converted to the oxidized form, whereas a small amount of reduced Trx1 remained even after prolonged treatment with higher Cr concentrations. Only one of the dithiols, presumably the active site, of Trx1 was oxidized by Cr(VI). Cr(VI) did not cause significant GSH depletion or oxidation indicating that Trx oxidation does not result from a general oxidation of cellular thiols. With purified Trx and thioredoxin reductase (TrxR) in vitro, Cr(VI) also resulted in Trx oxidation. It was determined that purified TrxR has pronounced Cr(VI) reducing activity, so competition for electron flow from TrxR might impair its ability to reduce Trx. The in vitro data also suggested some direct redox interaction between Cr(VI) and Trx. The ability of Cr(VI) to cause Trx oxidation in cells could contribute to its cytotoxic effects, and could have important implications for cell survival, redox-sensitive cell signaling, and the cells' tolerance of other oxidant insults. PMID:18328613

  12. Clarifying CB2 receptor-dependent and independent effects of THC on human lung epithelial cells

    SciTech Connect

    Sarafian, Theodore; Montes, Cindy; Harui, Airi

    Marijuana smoking is associated with a number of abnormal findings in the lungs of habitual smokers. Previous studies revealed that {delta}{sup 9}-tetrahydrocannabinol (THC) caused mitochondrial injury in primary lung epithelial cells and in the cell line, A549 [Sarafian, T. A., Kouyoumjian, S., Khoshaghideh, F., Tashkin, D. P., and Roth, M. D. (2003). Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cell energetics. Am J Physiol Lung Cell Mol Physiol 284, L298-306; Sarafian, T., Habib, N., Mao, J. T., Tsu, I. H., Yamamoto, M. L., Hsu, E., Tashkin, D. P., and Roth, M. D. (2005). Gene expression changes in human small airway epithelialmore » cells exposed to Delta9-tetrahydrocannabinol. Toxicol Lett 158, 95-107]. The role of cannabinoid receptors in this injury was unclear, as was the potential impact on cell function. In order to investigate these questions, A549 cells were engineered to over-express the type 2 cannabinoid receptor (CB2R) using a self-inactivating lentiviral vector. This transduction resulted in a 60-fold increase in CB2R mRNA relative to cells transduced with a control vector. Transduced cell lines were used to study the effects of THC on chemotactic activity and mitochondrial function. Chemotaxis in response to a 10% serum gradient was suppressed in a concentration-dependent manner by exposure to THC. CB2R-transduced cells exhibited less intrinsic chemotactic activity (p < 0.05) and were 80- to 100-fold more sensitive to the inhibitory effects of THC. Studies using SR144528, a selective CB2R antagonist, verified that these effects were mediated by the CB2R. Marijuana smoke extract, but not smoke extracts from tobacco or placebo marijuana cigarettes, reproduced these effects (p < 0.05). THC decreased ATP level and mitochondrial membrane potential ({psi}{sub m}) in both control and CB2R-transduced cells. However, these decreases did not play a significant role in chemotaxis inhibition since cyclosporine A, which protected against

  13. Interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in human epithelial cancer cells.

    PubMed

    Piyush, Tushar; Chacko, Anisha R; Sindrewicz, Paulina; Hilkens, John; Rhodes, Jonathan M; Yu, Lu-Gang

    2017-11-01

    Epidermal growth factor receptor (EGFR) is an important regulator of epithelial cell growth and survival in normal and cancerous tissues and is a principal therapeutic target for cancer treatment. EGFR is associated in epithelial cells with the heavily glycosylated transmembrane mucin protein MUC1, a natural ligand of galectin-3 that is overexpressed in cancer. This study reveals that the expression of cell surface MUC1 is a critical enhancer of EGF-induced EGFR activation in human breast and colon cancer cells. Both the MUC1 extracellular and intracellular domains are involved in EGFR activation but the predominant influence comes from its extracellular domain. Binding of galectin-3 to the MUC1 extracellular domain induces MUC1 cell surface polarization and increases MUC1-EGFR association. This leads to a rapid increase of EGFR homo-/hetero-dimerization and subsequently increased, and also prolonged, EGFR activation and signalling. This effect requires both the galectin-3 C-terminal carbohydrate recognition domain and its N-terminal ligand multi-merization domain. Thus, interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in epithelial cancer cells. As MUC1 and galectin-3 are both commonly overexpressed in most types of epithelial cancers, their interaction and impact on EGFR activation likely makes important contribution to EGFR-associated tumorigenesis and cancer progression and may also influence the effectiveness of EGFR-targeted cancer therapy.

  14. Human bronchial epithelial cells injury and cytokine production induced by Tityus serrulatus scorpion venom: An in vitro study.

    PubMed

    Rigoni, Vera Lucia Silva; Kwasniewski, Fabio H; Vieira, Rodolfo Paula; Linhares, Ingrid Sestrem; da Silva, Joelmir Lucena Veiga; Nogueira-Pedro, Amanda; Zamuner, Stella Regina

    2016-09-15

    Tityus serrulatus is the scorpion specie responsible for the majority of scorpion sting accidents in Brazil. Symptoms of envenomation by Tityus serrulatus range from local pain to severe systemic reactions such as cardiac dysfunction and pulmonary edema. Thus, this study has evaluated the participation of bronchial epithelial cells in the pulmonary effects of Tityus serrulatus scorpion venom (Tsv). Human bronchial epithelial cell line BEAS-2B were utilized as a model target and were incubated with Tsv (10 or 50 μg/mL) for 1, 3, 6 and 24 h. Effects on cellular response of venom-induce cytotoxicity were examined including cell viability, cell integrity, cell morphology, apoptosis/necrosis as well as cell activation through the release of pro-inflammatory cytokines IL-1β, IL-6 and IL-8. Tsv caused a decrease in cell viability at 10 and 50 μg/mL, which was confirmed by lactate dehydrogenase (LDH) measurement. Flow cytometry analyses revealed necrosis as the main cell death pathway caused by Tsv. Furthermore, Tsv induced the release of IL-1β, IL-6 and IL-8. Altogether, these results demonstrate that Tsv induces cytotoxic effects on bronchial epithelial cells, involving necrosis and release of pro-inflammatory cytokines, suggesting that bronchial epithelial cells may play a role in the pulmonary injury caused by Tsv. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Group B Streptococcus serotypes III and V induce apoptosis and necrosis of human epithelial A549 cells.

    PubMed

    Da Costa, Andréia Ferreira Eduardo; Pereira, Camila Serva; Santos, Gabriela Da Silva; Carvalho, Técia Maria Ulisses; Hirata, Raphael; De Mattos-Guaraldi, Ana Luiza; Rosa, Ana Cláudia De Paula; Nagao, Prescilla Emy

    2011-05-01

    Although group B Streptococcus (GBS) has been classically described as an exclusively extracellular pathogen, growing evidence suggests that it may be internalized by epithelial cells. However, the fates of intracellular GBS and of infected respiratory epithelial cells remain unclear. Little is known about the bacterial components involved in these processes. The present study investigated the bacterial internalization by A549 cells and the apoptosis/necrosis of the infected human epithelial cells. The morphological changes in A549 cells observed from 2 h post-infection with GBS included vacuolization and the formation of apoptotic bodies. Flow cytometry revealed that 81.2% of apoptotic A549 cells were infected with GBS serotype III 90356-liquor. Moreover, a double-staining assay using propidium iodide (PI)/Annexin V (AV) gave information about the numbers of viable (PI-/AV-) (18.27%) vs. early apoptotic (PI-/AV+) (73.83%) and late apoptotic cells (PI+/AV+) (7.37%) during infection of A549 cells with GBS III 90356-liquor. In addition, 37% necrotic cells were observed in A549 cells infected with GBS serotype V 90186-blood. In conclusion, GBS serotypes III and V induce apoptosis of epithelial cells in the early stages of GBS infection, resulting in tissue destruction, bacterial spreading and, in consequence, invasive disease or systemic infection.

  16. E-Cigarette Vapor Induces an Apoptotic Response in Human Gingival Epithelial Cells Through the Caspase-3 Pathway.

    PubMed

    Rouabhia, Mahmoud; Park, Hyun Jin; Semlali, Abdelhabib; Zakrzewski, Andrew; Chmielewski, Witold; Chakir, Jamila

    2017-06-01

    Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539-1547, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Bioaerosols from a Food Waste Composting Plant Affect Human Airway Epithelial Cell Remodeling Genes

    PubMed Central

    Chang, Ming-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

    2013-01-01

    The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 102 conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5–10 μm) having higher endotoxin levels than did fine particles (0.5–2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers. PMID:24368426

  18. Functional activity of L-carnitine transporters in human airway epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2016-02-01

    Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km<2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 μM) and a low affinity (Km>1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2015-07-01

    Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Genetic damage induced by organic extract of coke oven emissions on human bronchial epithelial cells.

    PubMed

    Zhai, Qingfeng; Duan, Huawei; Wang, Yadong; Huang, Chuanfeng; Niu, Yong; Dai, Yufei; Bin, Ping; Liu, Qingjun; Chen, Wen; Ma, Junxiang; Zheng, Yuxin

    2012-08-01

    Coke oven emissions are known as human carcinogen, which is a complex mixture of polycyclic aromatic hydrocarbon. In this study, we aimed to clarify the mechanism of action of coke oven emissions induced carcinogenesis and to identify biomarkers of early biological effects in a human bronchial epithelial cell line with CYP1A1 activity (HBE-CYP1A1). Particulate matter was collected in the oven area on glass filter, extracted and analyzed by GC/MS. DNA breaks and oxidative damage were evaluated by alkaline and endonucleases (FPG, hOGG1 and ENDO III)-modified comet assays. Cytotoxicity and chromosomal damage were assessed by the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The cells were treated with organic extract of coke oven emissions (OE-COE) representing 5, 10, 20, 40μg/mL extract for 24h. We found that there was a dose-effect relationship between the OE-COE and the direct DNA damage presented by tail length, tail intensity and Olive tail moment in the comet assay. The presence of lesion-specific endonucleases in the assays increased DNA migration after OE-COE treatment when compared to those without enzymes, which indicated that OE-COE produced oxidative damage at the level of pyrimidine and purine bases. The dose-dependent increase of micronuclei, nucleoplasmic bridges and nuclear buds in exposed cells was significant, indicating chromosomal and genomic damage induced by OE-COE. Based on the cytotoxic biomarkers in CBMN-Cyt assay, OE-COE may inhibit nuclear division, interfere with apoptosis, or induce cell necrosis. This study indicates that OE-COE exposure can induce DNA breaks/oxidative damage and genomic instability in HBE-CYP1A1 cells. The FPG-comet assay appears more specific for detecting oxidative DNA damage induced by complex mixtures of genotoxic substances. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Bioaerosols from a food waste composting plant affect human airway epithelial cell remodeling genes.

    PubMed

    Chang, Min-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

    2013-12-24

    The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 10(2) conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5-10 μm) having higher endotoxin levels than did fine particles (0.5-2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21 WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers.

  2. Differential Gene Expression in Normal Human Mammary Epithelial Cells Treated with Malathion Monitored by DNA Microarrays

    PubMed Central

    Gwinn, Maureen R.; Whipkey, Diana L.; Tennant, Lora B.; Weston, Ainsley

    2005-01-01

    Organophosphate pesticides are a major source of occupational exposure in the United States. Moreover, malathion has been sprayed over major urban populations in an effort to control mosquitoes carrying West Nile virus. Previous research, reviewed by the U.S. Environmental Protection Agency, on the genotoxicity and carcinogenicity of malathion has been inconclusive, although malathion is a known endocrine disruptor. Here, interindividual variations and commonality of gene expression signatures have been studied in normal human mammary epithelial cells from four women undergoing reduction mammoplasty. The cell strains were obtained from the discarded tissues through the Cooperative Human Tissue Network (sponsors: National Cancer Institute and National Disease Research Interchange). Interindividual variation of gene expression patterns in response to malathion was observed in various clustering patterns for the four cell strains. Further clustering identified three genes with increased expression after treatment in all four cell strains. These genes were two aldo–keto reductases (AKR1C1 and AKR1C2) and an estrogen-responsive gene (EBBP). Decreased expression of six RNA species was seen at various time points in all cell strains analyzed: plasminogen activator (PLAT), centromere protein F (CPF), replication factor C (RFC3), thymidylate synthetase (TYMS), a putative mitotic checkpoint kinase (BUB1), and a gene of unknown function (GenBank accession no. AI859865). Expression changes in all these genes, detected by DNA microarrays, have been verified by real-time polymerase chain reaction. Differential changes in expression of these genes may yield biomarkers that provide insight into interindividual variation in malathion toxicity. PMID:16079077

  3. The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells

    PubMed Central

    Richmond, Amy L.; Kabi, Amrita; Homer, Craig R.; García, Noemí Marina; Nickerson, Kourtney P.; NesvizhskiI, Alexey I.; Sreekumar, Arun; Chinnaiyan, Arul M.; Nuñez, Gabriel; McDonald, Christine

    2013-01-01

    BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn’s disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD. PMID:22387394

  4. Growth and differentiation of human lens epithelial cells in vitro on matrix

    NASA Technical Reports Server (NTRS)

    Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.; Aragon, G.; Lin, S. P.; Lui, G.; Polansky, J. R.

    2000-01-01

    PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

  5. Early establishment of epithelial apoptosis in the developing human small intestine.

    PubMed

    Vachon, P H; Cardin, E; Harnois, C; Reed, J C; Vézina, A

    2000-12-01

    In the adult small intestine, the dynamic renewal of the epithelium is characterized by a sequence of cell production in the crypts, cell maturation and cell migration to the tip of villi, where apoptosis is undertaken. Little is known about enterocytic apoptosis during development. In man, intestinal architectural features and functions are acquired largely by mid-gestation (18-20 wks); the question whether the establishment of enterocytic apoptotic processes parallels or not the acquisition of other intestinal functional features remains open. In the present study, we approached this question by examining enterocytic apoptosis during development of the human jejunum (9-20 wks gestation), using the ISEL (in situ terminal uridine deoxynucleotidyl nick-end labelling) method. Between 9 and 17 wks, apoptotic enterocytes were not evidenced. However, beginning at the 18 wks stage, ISEL-positive enterocytes were regularly observed at the tip of villi. Since the Bcl-2 family of proteins constitutes a critical checkpoint in apoptosis, acting upstream of the apoptotic machinery, we investigated the expression of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1). By immunofluorescence, we found that all homologs analyzed were expressed by enterocytes between 9 and 20 wks. However, Bcl-2 homologs underwent a gradual compartmentalization of epithelial expression along the maturing crypt-villus axis, to establish gradients of expression by 18-20 wks. Western blot analyses indicated that the expression levels of Bcl-2 homologs were modulated during morphogenesis of the crypt-villus axis, in parallel to their gradual compartmentalization of expression. Altogether, these data suggest that regulatory mechanisms of human enterocytic apoptosis become established by mid-gestation (18-20 wks) and coincide with the maturation of the crypt-villus axis of cell proliferation, differentiation and renewal.

  6. Effects of Platinum Nanocolloid in Combination with Gamma Irradiation on Normal Human Esophageal Epithelial Cells.

    PubMed

    Li, Qiang; Tanaka, Yoshiharu; Saitoh, Yasukazu; Miwa, Nobuhiko

    2016-05-01

    Our previous study demonstrated that platinum nanocolloid (Pt-nc), combined with lower-dose gamma irradiation at 3, 5, and 7 Gy significantly decreased proliferation and accelerated apoptosis of the human esophageal squamous cell carcinoma-derived cell line KYSE-70. The aim of the present study was to determine, under the same conditions as our previous study where gamma rays combined with Pt-nc were carcinostatic to KYSE-70 cells, if we could induce a radioprotective or the radiation-sensitizing effect on the human normal esophageal epithelial cells (HEEpiC). HEEpiC were treated with various Pt-nc concentrations and then irradiated with various gamma-ray doses. The proliferative status of HEEpiC was evaluated using trypan blue dye-exclusion and WST-8 assays. The cellular and nucleic morphological features were determined using crystal violet and Hoechst 33342 stainings, respectively. The intracellular level of reactive oxygen species (ROS) in HEEpiC was evaluated with a nitro blue tetrazolium (NBT) assay. The apoptotic status was detected with caspase-3, Bax, and Bcl-2 by Western blotting. Either Pt-nc or gamma irradiation could inhibit the growth of HEEpiC; however, their combined use exerted a significant proliferation-inhibitory effect in a Pt-nc dose-dependent manner than gamma irradiation alone. Pt-nc resulted in radiation sensitization rather than radiation protection on HEEpiC in vitro similar to KYSE-70 cells, when Pt-nc was administrated alone or combined with gamma irradiation. Thus, Pt-nc has an inhibitory effect on cell proliferation, a facilitative effect on apoptosis, and a certain degree of toxicity against HEEpiC.

  7. 4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells

    SciTech Connect

    Cheng Yahsin; Chang, Louis W.; Cheng Lichuan

    2007-05-01

    Epidemiological studies indicated that people exposed to dioxins were prone to the development of lung diseases including lung cancer. Animal studies demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased liver tumors and promoted lung metaplasia in females. Metabolic changes in 17{beta}-estradiol (E{sub 2}) resulted from an interaction between TCDD and E{sub 2} could be associated with gender difference. Previously, we reported that methoxylestradiols (MeOE{sub 2}), especially 4-MeOE{sub 2}, accumulated in human lung cells (BEAS-2B) co-treated with TCDD and E{sub 2}. In the present study, we demonstrate unique accumulation of 4-MeOE{sub 2}, as a result of TCDD/E{sub 2} interaction and revealed its bioactivity inmore » human lung epithelial cell line (H1355). 4-Methoxyestradiol treatment significantly decreased cell growth and increased mitotic index. Elevation of ROS and SOD activity, with a concomitant decrease in the intracellular GSH/GSSG ratio, was also detected in 4-MeOE{sub 2}-treated cells. Quantitative comet assay showed increased oxidative DNA damage in the 4-MeOE{sub 2}-treated H1355 cells, which could be significantly reduced by the anti-oxidant N-acetylcysteine (NAC). However, inhibition of cell growth and increase in mitotic arrest induced by 4-MeOE{sub 2} were unaffected by NAC. We concluded that 4-MeOE{sub 2} accumulation resulting from TCDD and E{sub 2} interaction would contribute to the higher vulnerability on lung pathogenesis in females when exposed to TCDD.« less

  8. Predicting the impact of diet and enzymopathies on human small intestinal epithelial cells

    PubMed Central

    Sahoo, Swagatika; Thiele, Ines

    2013-01-01

    Small intestinal epithelial cells (sIECs) have a significant share in whole body metabolism as they perform enzymatic digestion and absorption of nutrients. Furthermore, the diet plays a key role in a number of complex diseases including obesity and diabetes. The impact of diet and altered genetic backgrounds on human metabolism may be studied by using computational modeling. A metabolic reconstruction of human sIECs was manually assembled using the literature. The resulting sIEC model was subjected to two different diets to obtain condition-specific metabolic models. Fifty defined metabolic tasks evaluated the functionalities of these models, along with the respective secretion profiles, which distinguished between impacts of different dietary regimes. Under the average American diet, the sIEC model resulted in higher secretion flux for metabolites implicated in metabolic syndrome. In addition, enzymopathies were analyzed in the context of the sIEC metabolism. Computed results were compared with reported gastrointestinal (GI) pathologies and biochemical defects as well as with biomarker patterns used in their diagnosis. Based on our simulations, we propose that (i) sIEC metabolism is perturbed by numerous enzymopathies, which can be used to study cellular adaptive mechanisms specific for such disorders, and in the identification of novel co-morbidities, (ii) porphyrias are associated with both heme synthesis and degradation and (iii) disturbed intestinal gamma-aminobutyric acid synthesis may be linked to neurological manifestations of various enzymopathies. Taken together, the sIEC model represents a comprehensive, biochemically accurate platform for studying the function of sIEC and their role in whole body metabolism. PMID:23492669

  9. Combustion products of 1,3-butadiene are cytotoxic and genotoxic to human bronchial epithelial cells.

    PubMed Central

    Catallo, W J; Kennedy, C H; Henk, W; Barker, S A; Grace, S C; Penn, A

    2001-01-01

    Adverse health effects of airborne toxicants, especially small respirable particles and their associated adsorbed chemicals, are of growing concern to health professionals, governmental agencies, and the general public. Areas rich in petrochemical processing facilities (e.g., eastern Texas and southern California) chronically have poor air quality. Atmospheric releases of products of incomplete combustion (e.g., soot) from these facilities are not subject to rigorous regulatory enforcement. Although soot can include respirable particles and carcinogens, the toxicologic and epidemiologic consequences of exposure to environmentally relevant complex soots have not been well investigated. Here we continue our physico-chemical analysis of butadiene soot and report effects of exposure to this soot on putative targets, normal human bronchial epithelial (NHBE) cells. We examined organic extracts of butadiene soot by gas chromatography-mass spectrometry (GC-MS), probe distillation MS, and liquid chromatography (LC)-MS-MS. Hundreds of aromatic hydrocarbons and polycyclic aromatic hydrocarbons with molecular mass as high as 1,000 atomic mass units were detected, including known and suspected human carcinogens (e.g., benzo(a)pyrene). Butadiene soot particles also had strong, solid-state free-radical character in electron spin resonance analysis. Spin-trapping studies indicated that fresh butadiene soot in a buffered aqueous solution containing dimethylsulfoxide (DMSO) oxidized the DMSO, leading to CH(3)* radical formation. Butadiene soot DMSO extract (BSDE)-exposed NHBE cells displayed extranuclear fluorescence within 4 hr of exposure. BSDE was cytotoxic to > 20% of the cells at 72 hr. Morphologic alterations, including cell swelling and membrane blebbing, were apparent within 24 hr of exposure. These alterations are characteristic of oncosis, an ischemia-induced form of cell death. BSDE treatment also produced significant genotoxicity, as indicated by binucleated cell

  10. Amplification and overexpression of aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells.

    PubMed

    Chung, C M; Man, C; Jin, Y; Jin, C; Guan, X Y; Wang, Q; Wan, T S K; Cheung, A L M; Tsao, S W

    2005-07-01

    Immortalization is an early and essential step of human carcinogenesis. Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers. We have previously reported that gain and amplification of chromosome 20q is a non-random and common event in immortalized human ovarian surface epithelial (HOSE) cells. The chromosome 20q harbors genes including TGIF2 (20q11.2-q12), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and AURKA (20q13.2-q13.3), which were previously reported to be amplified and overexpressed in ovarian cancers. Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium. Investigation of the involvement of these genes was examined in four pairs of pre-crisis (preimmortalized) and post-crisis (immortalized) HOSE cells. Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time-quantitative polymerase chain reaction (RT-QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined. Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems. The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells. Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q13.2-13.3) where AURKA resides. DNA amplification of AURKA was also confirmed using semi-quantitative PCR. Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis. Copyright (c) 2005 Wiley-Liss, Inc.

  11. Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

    SciTech Connect

    Montoudis, Alain; Delvin, Edgard; Canadian Institute of Health Research, Group of the Functional Development and Physiopathology of the Digestive Tract, and Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, Que., Canada J1H 5N4

    2006-01-06

    Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferationmore » and viability. Studies using these transfected cells cultured with [{sup 14}C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [{sup 35}S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.« less

  12. Pirfenidone may revert the epithelial-to-mesenchymal transition in human lung adenocarcinoma.

    PubMed

    Kurimoto, Ryota; Ebata, Takahiro; Iwasawa, Shunichiro; Ishiwata, Tsukasa; Tada, Yuji; Tatsumi, Koichiro; Takiguchi, Yuichi

    2017-07-01

    The epithelial-to-mesenchymal transition (EMT) in cancer is associated with invasion, metastasis and chemoresistance. Recent studies have revealed the increased expression of programmed death-ligand 1 (PD-L1) in cells undergoing EMT. The underlying mechanism of EMT involves transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2). Pirfenidone and the known EMT-suppressor nintedanib suppress pulmonary fibrosis partially through suppression of TGF-β. The present study aimed to determine whether pirfenidone has the potential to induce EMT-reversion, using nintedanib as a reference. The human lung adenocarcinoma cell lines A-549, HCC-827, and PC-9 were treated with TGF-β and FGF-2 to induce EMT. The EMT-induced cells were further treated with pirfenidone or nintedanib. Phenotypic alterations associated with EMT were assessed by examining the following: i) The expression levels of E-cadherin, vimentin, fibronectin and slug, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescent immunohistochemistry; ii) cell motility via wound-healing assays; and iii) the expression of PD-L1 using RT-qPCR. The combination of TGF-β and FGF-2 successfully induced EMT in all three cell lines, characterized by a significant reduction in E-cadherin expression in the A-549 and HCC-827 cells, increased expression levels of vimentin, fibronectin, slug and PD-L1, and increased cell motility in all three cell lines. Pirfenidone and nintedanib reverted all of these phenotypes, with the exception of unaltered E-cadherin expression in all three cell lines, and inconsistent expression of vimentin in the HCC-827 and PC-9 cells. Thus, pirfenidone and nintedanib have the ability to induce EMT-reversion in human lung adenocarcinoma.

  13. Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

    SciTech Connect

    Li Hongzhen; Zhou Jianjun; Miki, Jun

    2008-01-01

    Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetalmore » bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.« less

  14. Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

    PubMed

    Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

    2017-02-28

    Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

  15. 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...

  16. Detergent solubilization of the EGF receptor from A431 cells

    NASA Technical Reports Server (NTRS)

    Dayanidhi, R.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Functional reconstitution of purified preparations of human epidermal growth factor receptor (EGFR) requires dissociation of the protein from its plasma membrane lipid environment. Solubilization of membrane proteins in this manner requires the use of detergents, which are known to disrupt plasma membrane lipid/protein interactions. We have investigated the ability of three nonionic detergents to solubilize the human EGFR selectively, and have also analyzed the effect of these various treatments on the intrinsic tyrosyl kinase activity of the receptor. The nonionic detergent known as n-octyl glucoside (n-octyl beta-D-glucopyranoside) was found to give the best combination of selectivity, yield, and maintenance of enzymatic activity of the human EGFR.

  17. Control of growth and squamous differentiation in normal human bronchial epithelial cells by chemical and biological modifiers and transferred genes.

    PubMed Central

    Pfeifer, A M; Lechner, J F; Masui, T; Reddel, R R; Mark, G E; Harris, C C

    1989-01-01

    The majority of human lung cancers arise from bronchial epithelial cells. The normal pseudostratified bronchial epithelium is composed of basal, mucous, and ciliated cells. This multi-differentiated epithelium usually responds to xenobiotics and physical injury by undergoing basal cell hyperplasia, mucous cell hyperplasia, and squamous metaplasia. One step of the multistage process of carcinogenesis is thought to involve aberrations in control of the squamous metaplastic processes. Decreased responsiveness to regulators of terminal squamous differentiation may confer a selective clonal expansion advantage to an initiated cell. We studied the effects of endogenous [e.g., transforming growth factor beta 1 (TGF-beta 1) and serum] and exogenous [e.g., 12-O-tetradecanoyl-13-phorbol-acetate (TPA), tobacco smoke condensate, and aldehydes] modifiers of normal human bronchial epithelial (NHBE) cell in a serum-free culture system. NHBE cells are growth inhibited by all of these compounds and induced to undergo squamous differentiation by TGF-beta 1 or TPA. In contrast, lung carcinoma cell lines are relatively resistant to inducers of terminal squamous differentiation which may provide them with a selective growth advantage. Chemical agents and activated protooncogenes (ras,raf,myc) altered the response to endogenous and exogenous inducers of squamous differentiation and caused extended cellular lifespan, aneuploidy, and/or tumorigenicity. The data suggest a close relationship between dysregulation of terminal differentiation pathways and neoplastic transformation of human bronchial epithelial cells. PMID:2538323

  18. Characterization of Epithelial Progenitors in Normal Human Palatine Tonsils and Their HPV16 E6/E7-Induced Perturbation

    PubMed Central

    Kang, Sung Yoon Catherine; Kannan, Nagarajan; Zhang, Lewei; Martinez, Victor; Rosin, Miriam P.; Eaves, Connie J.

    2015-01-01

    Summary Human palatine tonsils are oropharyngeal lymphoid tissues containing multiple invaginations (crypts) in which the continuity of the outer surface epithelium is disrupted and the isolated epithelial cells intermingle with other cell types. We now show that primitive epithelial cells detectable in vitro in 2D colony assays and in a 3D culture system are CD44+NGFR+ and present in both surface and crypt regions. Transcriptome analysis indicated a high similarity between CD44+NGFR+ cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. Lentiviral transduction of CD44+NGFR+ cells from both regions with human papillomavirus 16-encoded E6/E7 prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Our findings therefore reveal a shared, site-independent, hierarchical organization, differentiation potential, and transcriptional profile of normal human tonsillar epithelial progenitor cells. They also introduce a new model for investigating the mechanisms of their transformation. PMID:26527383

  19. Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

    SciTech Connect

    Jang, Soo Hwa; Choi, Changsun; Hong, Seong-Geun

    2009-06-26

    Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a rolemore » in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.« less

  20. Transcriptional program of ciliated epithelial cells reveals new cilium and centrosome components and links to human disease.

    PubMed

    Hoh, Ramona A; Stowe, Timothy R; Turk, Erin; Stearns, Tim

    2012-01-01

    Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease.

  1. Transcriptional Program of Ciliated Epithelial Cells Reveals New Cilium and Centrosome Components and Links to Human Disease

    PubMed Central

    Hoh, Ramona A.; Stowe, Timothy R.; Turk, Erin; Stearns, Tim

    2012-01-01

    Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease. PMID:23300604

  2. Differential Gene Expression Profiling of Functionally and Developmentally Distinct Human Prostate Epithelial Populations

    PubMed Central

    Liu, Haibo; Cadaneanu, Radu M; Lai, Kevin; Zhang, Baohui; Huo, Lihong; An, Dong Sun; Li, Xinmin; Lewis, Michael S; Garraway, Isla P

    2015-01-01

    BACKGROUND Human fetal prostate buds appear in the 10th gestational week as solid cords, which branch and form lumens in response to androgen 1. Previous in vivo analysis of prostate epithelia isolated from benign prostatectomy specimens indicated that Epcam+CD44−CD49fHi basal cells possess efficient tubule initiation capability relative to other subpopulations 2. Stromal interactions and branching morphogenesis displayed by adult tubule-initiating cells (TIC) are reminiscent of fetal prostate development. In the current study, we evaluated in vivo tubule initiation by human fetal prostate cells and determined expression profiles of fetal and adult epithelial subpopulations in an effort to identify pathways used by TIC. METHODS Immunostaining and FACS analysis based on Epcam, CD44, and CD49f expression demonstrated the majority (99.9%) of fetal prostate epithelial cells (FC) were Epcam+CD44− with variable levels of CD49f expression. Fetal populations isolated via cell sorting were implanted into immunocompromised mice. Total RNA isolation from Epcam+CD44−CD49fHi FC, adult Epcam+CD44−CD49fHi TIC, Epcam+CD44+CD49fHi basal cells (BC), and Epcam+CD44−CD49fLo luminal cells (LC) was performed, followed by microarray analysis of 19 samples using the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data was analyzed using Partek Genomics Suite Version 6.4. Genes selected showed >2-fold difference in expression and P < 5.00E-2. Results were validated with RT-PCR. RESULTS Grafts retrieved from Epcam+CD44− fetal cell implants displayed tubule formation with differentiation into basal and luminal compartments, while only stromal outgrowths were recovered from Epcam- fetal cell implants. Hierarchical clustering revealed four distinct groups determined by antigenic profile (TIC, BC, LC) and developmental stage (FC). TIC and BC displayed basal gene expression profiles, while LC expressed secretory genes. FC had a unique profile with the most similarities to adult TIC

  3. PD-L1 Expression Promotes Epithelial to Mesenchymal Transition in Human Esophageal Cancer.

    PubMed

    Chen, Lujun; Xiong, Yuqi; Li, Jing; Zheng, Xiao; Zhou, Qi; Turner, Abbey; Wu, Changping; Lu, Binfeng; Jiang, Jingting

    2017-01-01

    PD-L1 (Programmed cell death 1 ligand 1, PD-L1), an essential immune checkpoint molecule in the tumor microenvironment, is an important target for cancer immunotherapy. We have previously reported that its expression in human gastric and esophageal cancer tissues is significantly associated with cancer progression and patients' postoperative prognoses. Its expression in cancer cells is well known to inhibit the T cell-mediated anti-tumor response, and this mechanism of action has been targeted for cancer immunotherapy. As of now, the autonomous effect of PD-L1 on cancer cells is not well understood, thus our present study aimed to examine the role of PD-L1 intervention in cellular biological functions, especially epithelial to mesenchymal transition (EMT), of the human esophageal cancer cell line, Eca-109 cells. Immunohistochemistry assay was used to investigate the correlation between expression of PD-L1 and EMT markers in human esophageal cancer tissues. Intervention of PD-L1 by using RNAi and over-expression methods were used to study the role of PD-L1 in regulation of biological behaviors and EMT in Eca-109 cells. Our clinical and pathological data demonstrated that tumor samples in the EMT positive subgroup had higher PD-L1 expression than those in the EMT negative subgroup. By manipulating PD-L1 expression in Eca-109 cells either through ablation or overexpression of wild type and the cytoplasmic domain-truncated mutant, we demonstrated that PD-L1 expression significantly promoted the cell viability, migration and EMT phenotype. Furthermore, our study also indicated that PD-1 fusion protein mediated stimulation of PD-L1 and the cytoplasmic domain of PD-L1 played a critical role in promoting EMT phenotype of Eca-109 cells, thereby suggesting that PD-1 receptor usually by triggering the reverse signaling can effect PD-L1 mediated regulation of esophageal cancer cell response. Our present study reveals a tumor cell-autonomous role of PD-L1 signaling in promoting

  4. Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells

    PubMed Central

    Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

    2017-01-01

    Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn’t differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication. PMID:28886142

  5. Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells

    PubMed Central

    Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G.; Kuemmerle, John F.; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I.

    2014-01-01

    Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA–depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. PMID:25143399

  6. Towards optimization of an organotypic assay system that imitates human hair follicle-like epithelial-mesenchymal interactions.

    PubMed

    Havlickova, B; Bíró, T; Mescalchin, A; Arenberger, P; Paus, R

    2004-10-01

    Human hair growth can currently be studied in vitro by the use of organ-cultured scalp hair follicles (HFs). However, simplified organotypic systems are needed for dissecting the underlying epithelial-mesenchymal interactions and as screening tools for candidate hair growth-modulatory agents. To optimize the design and culture conditions of previously published organotypic systems that imitate epithelial-mesenchymal interactions in the human HF as closely as possible. Continuous submerged organotypic 'sandwich' cultures were established. These consist of a pseudodermis (collagen I mixed with and contracted by human interfollicular dermal fibroblasts) on which one of two upper layers is placed: either a mixture of Matrigel basement membrane matrix (BD Biosciences, Bedford, MA, U.S.A.) and follicular dermal papilla fibroblasts (DPC), with outer root sheath keratinocytes (ORSK) layered on the top ('layered' system), or a mixture of Matrigel, DPC and ORSK ('mixed' system). Morphological and functional characteristics of these 'folliculoid sandwiches' were then assessed by routine histology, histomorphometry and immunohistochemistry. In both 'layered' and 'mixed' systems, the ORSK formed spheroid epithelial cell aggregates, which retained their characteristic keratin expression pattern (i.e. cytokeratin 6). In the 'mixed' sandwich model the size of the epithelial cell aggregates was smaller, but the numbers of ORSK were significantly higher than in the 'layered' model at day 14 in the culture. ORSK proliferated better in the 'mixed' than in the 'layered' sandwich system, regardless of the calcium or serum content of the media, whereas apoptosis of ORSK was lowest in the 'mixed' system in serum-free, low calcium medium. The kinetics of proliferation and apoptosis of DPC, which retained their characteristic expression of versican, were similar in both systems. However, proliferation and apoptosis of DPC were higher in the presence of serum and/or under high calcium

  7. Does human endometrial LGR5 gene expression suggest the existence of another hormonally regulated epithelial stem cell niche?

    PubMed

    Tempest, N; Baker, A M; Wright, N A; Hapangama, D K

    2018-06-01

    Is human endometrial leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) gene expression limited to the postulated epithelial stem cell niche, stratum basalis glands, and is it hormonally regulated? LGR5 expressing cells are not limited to the postulated stem cell niche but LGR5 expression is hormonally regulated. The human endometrium is a highly regenerative tissue; however, endometrial epithelial stem cell markers are yet to be confirmed. LGR5 is a marker of stem cells in various epithelia. The study was conducted at a University Research Institute. Endometrial samples from 50 healthy women undergoing benign gynaecological surgery with no endometrial pathology at the Liverpool Women's hospital were included and analysed in the following six sub-categories; proliferative, secretory phases of menstrual cycle, postmenopausal, those using oral and local progestagens and samples for in vitro explant culture. In this study, we used the gold standard method, in situ hybridisation (ISH) along with qPCR and a systems biology approach to study the location of LGR5 gene expression in full thickness human endometrium and Fallopian tubes. The progesterone regulation of endometrial LGR5 was examined in vivo and in short-term cultured endometrial tissue explants in vitro. LGR5 expression was correlated with epithelial proliferation (Ki67), and expression of previously reported epithelia progenitor markers (SOX9 and SSEA-1) immunohistochemistry (IHC). LGR5 gene expression was significantly higher in the endometrial luminal epithelium than in all other epithelial compartments in the healthy human endometrium, including the endometrial stratum basalis (P < 0.05). The strongest SSEA-1 and SOX9 staining was observed in the stratum basalis glands, but the general trend of SOX9 and SSEA-1 expression followed the same cyclical pattern of expression as LGR5. Stratum functionalis epithelial Ki67-LI and LGR5 expression levels correlated significantly (r = 0.74, P = 0

  8. Human papillomavirus-32-associated focal epithelial hyperplasia accompanying HPV-16-positive papilloma-like lesions in oral mucosa.

    PubMed

    Liu, Na; Wang, Jiayi; Lei, Lei; Li, Yanzhong; Zhou, Min; Dan, Hongxia; Zeng, Xin; Chen, Qianming

    2013-05-01

    Human papillomavirus infection can cause a variety of benign or malignant oral lesions, and the various genotypes can cause distinct types of lesions. To our best knowledge, there has been no report of 2 different human papillomavirus-related oral lesions in different oral sites in the same patient before. This paper reported a patient with 2 different oral lesions which were clinically and histologically in accord with focal epithelial hyperplasia and oral papilloma, respectively. Using DNA extracted from these 2 different lesions, tissue blocks were tested for presence of human papillomavirus followed by specific polymerase chain reaction testing for 6, 11, 13, 16, 18, and 32 subtypes in order to confirm the clinical diagnosis. Finally, human papillomavirus-32-positive focal epithelial hyperplasia accompanying human papillomavirus-16-positive oral papilloma-like lesions were detected in different sites of the oral mucosa. Nucleotide sequence sequencing further confirmed the results. So in our clinical work, if the simultaneous occurrences of different human papillomavirus associated lesions are suspected, the multiple biopsies from different lesions and detection of human papillomavirus genotype are needed to confirm the diagnosis.

  9. Continual exposure to cigarette smoke extracts induces tumor-like transformation of human nontumor bronchial epithelial cells in a microfluidic chip.

    PubMed

    Li, Encheng; Xu, Zhiyun; Liu, Fen; Wang, Huiling; Wen, Jiabin; Shao, Shujuan; Zhang, Lichuan; Wang, Lei; Liu, Chong; Lu, Jianxin; Wang, Wenxin; Gao, Zhancheng; Wang, Qi

    2014-08-01

    Heavy cigarette smoking-related chronic obstructive pulmonary disease is an independent risk factor for lung squamous carcinoma. However, the mechanisms underlying the malignant transformation of bronchial epithelial cells are unclear. In our study, human tumor-adjacent bronchial epithelial cells were obtained from 10 cases with smoking-related chronic obstructive pulmonary disease and lung squamous carcinoma and cultured in an established microfluidic chip for continual exposure to cigarette smoke extracts (CSE) to investigate the potential tumor-like transformation and mechanisms. The integrated microfluidic chip included upstream concentration gradient generator and downstream cell culture chambers supplied by flowing medium containing different concentrations of CSE. Our results showed that continual exposure to low doses of CSE promoted cell proliferation whereas to high doses of CSE triggered cell apoptosis. Continual exposure to CSE promoted reactive oxygen species production in human epithelial cells in a dose-dependent manner. More importantly, continual exposure to low dose of CSE promoted the epithelial-to-mesenchymal transition process and anchorage-independent growth, and increased chromosome instability in bronchial epithelial cells, accompanied by activating the GRP78, NF-κB, and PI3K pathways. The established microfluidic chip is suitable for primary culture of human tumor-adjacent bronchial epithelial cells to investigate the malignant transformation. Continual exposure to low doses of CSE promoted tumor-like transformation of human nontumor bronchial epithelial cells by inducing reactive oxygen species production and activating the relevant signaling.

  10. Reactivation of NCAM1 defines a subpopulation of human adult kidney epithelial cells with clonogenic and stem/progenitor properties.

    PubMed

    Buzhor, Ella; Omer, Dorit; Harari-Steinberg, Orit; Dotan, Zohar; Vax, Einav; Pri-Chen, Sara; Metsuyanim, Sally; Pleniceanu, Oren; Goldstein, Ronald S; Dekel, Benjamin

    2013-11-01

    The nephron is composed of a monolayer of epithelial cells that make up its various compartments. In development, these cells begin as mesenchyme. NCAM1, abundant in the mesenchyme and early nephron lineage, ceases to express in mature kidney epithelia. We show that, once placed in culture and released from quiescence, adult human kidney epithelial cells (hKEpCs), uniformly positive for CD24/CD133, re-express NCAM1 in a specific cell subset that attains a stem/progenitor state. Immunosorted NCAM1(+) cells overexpressed early nephron progenitor markers (PAX2, SALL1, SIX2, WT1) and acquired a mesenchymal fate, indicated by high vimentim and reduced E-cadherin levels. Gene expression and microarray analysis disclosed both a proximal tubular origin of these cells and molecules regulating epithelial-mesenchymal transition. NCAM1(+) cells generated clonal progeny when cultured in the presence of fetal kidney conditioned medium, differentiated along mesenchymal lineages but retained the unique propensity to generate epithelial kidney spheres and produce epithelial renal tissue on single-cell grafting in chick CAM and mouse. Depletion of NCAM1(+) cells from hKEpCs abrogated stemness traits in vitro. Eliminating these cells during the regenerative response that follows glycerol-induced acute tubular necrosis worsened peak renal injury in vivo. Thus, higher clone-forming and developmental capacities characterize a distinct subset of adult kidney-derived cells. The ability to influence an endogenous regenerative response via NCAM1 targeting may lead to novel therapeutics for renal diseases. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  11. cAMP-dependent activation of protein kinase A attenuates respiratory syncytial virus-induced human airway epithelial barrier disruption

    PubMed Central

    Harford, Terri J.; Linfield, Debra T.; Altawallbeh, Ghaith; Midura, Ronald J.; Ivanov, Andrei I.; Piedimonte, Giovanni

    2017-01-01

    Airway epithelium forms a barrier to the outside world and has a crucial role in susceptibility to viral infections. Cyclic adenosine monophosphate (cAMP) is an important second messenger acting via two intracellular signaling molecules: protein kinase A (PKA) and the guanidine nucleotide exchange factor, Epac. We sought to investigate effects of increased cAMP level on the disruption of model airway epithelial barrier caused by RSV infection and the molecular mechanisms underlying cAMP actions. Human bronchial epithelial cells were infected with RSV-A2 and treated with either cAMP releasing agent, forskolin, or cAMP analogs. Structure and functions of the Apical Junctional Complex (AJC) were evaluated by measuring transepithelial electrical resistance and permeability to FITC-dextran, and determining localization of AJC p