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Sample records for a431 human epithelial

  1. Pheophorbide a mediated photodynamic therapy against human epidermoid carcinoma cells (A431)

    NASA Astrophysics Data System (ADS)

    Chen, Yi-Chun; Li, Wen-Tyng

    2011-02-01

    The objective of this study was to characterize the death mechanism of human epidermoid carcinoma cells (A431) triggered by photodynamic therapy (PDT) with pheophorbide a. First of all, significant inhibition on the survival of A431 cells (< 20 %) was observed when an irradiation dose of 5.1 J/cm2 combined with 125 ng/ml of pheophorbide a was applied. Survival rate of human keratinocyte cells was over 70 % under the same PDT parameters, suggesting that pheophorbide a killed cancer cells selectively. Mitochondria were the main target sites where pheophorbide a accumulated. Formation of reactive oxygen species (ROS) was detected after PDT. Addition of antioxidant N-Acetyl cysteine prevented ROS production and increased cell survival thereafter. The decrease in cellular ATP level was also observed at 6 hrs after PDT. Typical apoptotic cellular morphology and a collapse of mitochondrial membrane potential occurred after PDT. The loss of mitochondrial membrane potential led to the release of cytochrome c from the mitochondria to the cytosol, followed by activation of caspase-9 and caspase-3. The activation of caspase-3 resulted in poly(ADP-ribose) polymerase (PARP) cleavage in A431 cells, followed by DNA fragmentation. In conclusion, the results demonstrated that pheophorbide a possessed photodynamic action against A431 cells, mainly through apoptosis mediated by mitochondrial intrinsic pathway triggered by ROS.

  2. Density-dependent induction of 92-kd type IV collagenase activity in cultures of A431 human epidermoid carcinoma cells.

    PubMed Central

    Xie, B.; Bucana, C. D.; Fidler, I. J.

    1994-01-01

    We examined the in vitro regulation of the production of two type IV collagenases, MMP-2 and MMP-9, by A431 human epidermoid carcinoma cells. The A431 cells were cultured under sparse or confluent conditions. The addition of transforming growth factor-beta (TGF-beta) or phorbolester-TPA to sparse cultures induced low levels of MMP-9 secretion, whereas in confluent cultures only TGF-beta produced this effect. Neither treatment altered the level of constitutive secretion of MMP-2. Treatment of sparse, actively growing cultures but not confluent stationary cultures with both TGF-beta and TPA produced synergistic induction of MMP-9 but did not affect MMP-2. A431 cells were grown as discrete large monolayer colonies. Radiolabeling with [3H]leucine or [3H]thymidine followed by autoradiography revealed that all the A431 cells in the colonies were metabolically active and only those on the periphery were dividing. Only these dividing A431 cells stained positive by anti-MMP-9 antibodies. Our results demonstrate that the synergistic induction of MMP-9 secretion in A431 cells occurs subsequent to stimulation by external signals in only noncontact-inhibited dividing tumor cells. These regulatory mechanisms may account for the in vivo finding that many proteinases are localized at the invasion front of a neoplasm where tumor cells are dividing and accessible to various environmental signals. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8178929

  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. Inhibitory effect of berberine on human skin squamous cell carcinoma A431 cells.

    PubMed

    Li, D X; Zhang, J; Zhang, Y; Zhao, P W; Yang, L M

    2015-09-08

    Berberine (BBR) is a natural alkaloid with significant anti-tumor activity against many types of cancer cells. In this study, we investigated the molecular mechanisms employed by BBR to repress the proliferation and growth of skin squamous cell carcinoma A431 cells. Berberine was reported to inhibit the proliferation of A431 cells in a dose- and time-dependent manner and was observed to induce a series of biochemical events, including the loss of mitochondrial membrane potential, release of cytochrome-c to cytosol, induction of proteins of the Bcl-2 family and caspases, and the cleavage of poly(ADP)-ribose polymerase. This suggested its ability to induce apoptosis. The results of a wound healing test revealed that berberine inhibited the migration of A431 cells. Ezrin was transfected into A431 cells by RNA interference. The level of expression of Ezrin in the transfected A431 cells was observed to decrease with berberine treatment, which suggested that berberine might inhibit the invasion of A431 cells through Ezrin. The results of this study demonstrated that berberine could potentially inhibit proliferation, induce apoptosis, and inhibit the invasion of A431 cells.

  5. Honokiol, a chemopreventive agent against skin cancer, induces cell cycle arrest and apoptosis in human epidermoid A431 cells.

    PubMed

    Chilampalli, Chandeshwari; Guillermo, Ruth; Kaushik, Radhey S; Young, Alan; Chandrasekher, Gudiseva; Fahmy, Hesham; Dwivedi, Chandradhar

    2011-11-01

    Honokiol is a plant lignan isolated from bark and seed cones of Magnolia officinalis. Recent studies from our laboratory indicated that honokiol pretreatment decreased ultraviolet B-induced skin cancer development in SKH-1 mice. The aim of the present investigation was to study the effects of honokiol on human epidermoid squamous carcinoma A431 cells and to elucidate possible mechanisms involved in preventing skin cancer. A431 cells were pretreated with different concentrations of honokiol for a specific time period and investigated for effects on apoptosis and cell cycle analysis. Treatment with honokiol significantly decreased cell viability and cell proliferation in a concentration- and time-dependent manner. Honokiol pretreatment at 50 μmol/L concentration induced G0/G1 cell cycle arrest significantly (P < 0.05) and decreased the percentage of cells in the S and G2/M phase. Honokiol down-regulated the expression of cyclin D1, cyclin D2, Cdk2, Cdk4 and Cdk6 proteins and up-regulated the expression of Cdk's inhibitor proteins p21 and p27. Pretreatment of A431 cells with honokiol leads to induction of apoptosis and DNA fragmentation. These findings indicate that honokiol provides its effects in squamous carcinoma cells by inducing cell cycle arrest at G0/G1 phase and apoptosis.

  6. 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; George, Jasmine; Ray, Ratan Singh; Hans, Rajendra K.; Prasad, Sahdeo; Shukla, Yogeshwer

    2009-06-26

    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 of 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.

  7. Several types of sodium-conducting channel in human carcinoma A-431 cells.

    PubMed

    Negulyaev YuA; Vedernikova, E A; Mozhayeva, G N

    1994-08-24

    Patch clamp method in outside-out configuration was used to search for cation channels which possibly mediate sodium influx through plasma membrane in A-431 carcinoma cells. We found four types of nonvoltage-gated Na-conducting channel. The first of 9-10 pS conductance (145 mM Na+, 30 degrees C) seems to be Na-selective; three others were characterized with conductance values of 24, 35 and 65 pS and lower selectivity among cations. Na-selective channels (9-10 pS) were not blocked by tetrodotoxin (1 microM). External application of amiloride (0.1-2 mM) resulted in a reversible inhibition of single currents through Na-selective channels.

  8. 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; George, Jasmine; Shukla, Yogeshwer

    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 of 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.

  9. 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

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

    2013-07-01

    Non-melanoma skin cancers (NMSCs), one of the most common neoplasms, cause 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 antiproliferative 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 fisetin (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 cytochrome c and Smac/DIABLO from mitochondria; (v) activation of caspases; and (vi) cleavage of Poly(ADP-ribose) polymerase (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.

  10. Hydroxyl radical (·OH) played a pivotal role in oridonin-induced apoptosis and autophagy in human epidermoid carcinoma A431 cells.

    PubMed

    Yu, Yang; Fan, Si Miao; Song, Jun Ke; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2012-01-01

    Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis through tyrosine kinase pathway. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431 cell as a test object. Besides apoptosis, oridonin also induced A431 cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431 cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, N-acetylcysteine (NAC) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (superoxide dismutase (SOD), catalase (CAT)) and non-enzyme antioxidants (glutathione (GSH)) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (·OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431 cells in a dose-dependent manner. In addition, after treatment with ·OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that ·OH played the pivotal role in oridonin induced apoptosis and autophagy in A431 cells.

  11. Involvement of retinoblastoma (Rb) and E2F transcription factors during photodynamic therapy of human epidermoid carcinoma cells A431.

    PubMed

    Ahmad, N; Gupta, S; Mukhtar, H

    1999-03-11

    Photodynamic therapy (PDT), a promising new therapeutic modality for the management of a variety of solid malignancies and many non-malignant diseases, is a bimodal therapy using a porphyrin based photosensitizing chemical and visible light. The proper understanding of the mechanism of PDT-mediated cancer cell-kill may result in improving the efficacy of this treatment modality. Earlier we have shown (Proc. Natl. Acad. Sci. USA; 95: 6977-6982, 1998) that silicon phthalocyanine (Pc4)-PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21 which, by inhibiting cyclins (E and D1) and cyclin dependent kinases (cdk2 and cdk6), results in a G0/G1-phase arrest followed by apoptosis in human epidermoid carcinoma cells A431. We have also demonstrated the generation of nitric oxide during PDT-mediated apoptosis (Cancer Res.; 58: 1785-1788, 1998). Retinoblastoma (pRb) and E2F family transcription factors are important proteins, which regulate the G1-->S transition in the cell cycle. Here, we provide evidence for the involvement of pRb-E2F/DP machinery as an important contributor of PDT-mediated cell cycle arrest and apoptosis. Western blot analysis demonstrated a decrease in the hyper-phosphorylated form of pRb at 3, 6 and 12 h post-PDT with a relative increase in hypo-phosphorylated pRb. Western blot analysis also revealed that PDT-caused decrease in phosphorylation of pRb occurs at serine-780. The ELISA data demonstrated a time dependent accumulation of hypo-phosphorylated pRb by PDT. This response was accompanied with down-regulation in the protein expression of all five E2F (1-5) family transcription factors, and their heterodimeric partners DP1 and DP2. These results suggest that Pc4-PDT of A431 cells results in a down regulation of hyper-phosphorylated pRb protein with a relative increase in hypo-phosphorylated pRb that, in turn, compromises with the availability of free E2F. We suggest that these events result in a stoppage of the cell cycle

  12. Recombinant human tumor necrosis factor alpha does not potentiate cell killing after photodynamic therapy with a silicon phthalocyanine in A431 human epidermoid carcinoma cells.

    PubMed

    Azizuddin, K; Kalka, K; Chiu, S M; Ahmad, N; Mukhtar, H; Separovic, D

    2001-02-01

    Photodynamic therapy (PDT) is a novel cancer treatment utilizing a photosensitizer, visible light and oxygen. PDT with the silicon phthalocyanine Pc 4, a new photosensitizer, is highly effective in cancer cell destruction and tumor ablation. The mechanisms underlying cancer cell killing by PDT are not fully understood. Tumor necrosis factor alpha (TNF) is a multifunctional cytokine that has been implicated in photocytotoxicity. We asked whether recombinant human TNF (rhTNF) affects Pc 4-PDT cytotoxicity in A431 human epidermoid carcinoma cells. Co-treatment of A431 cells with various doses of Pc 4-PDT and a sub-lethal rhTNF dose led to a sub-additive reduction in cell survival. In addition, in the presence of Pc 4-PDT or rhTNF, caspase-3 activity and apoptosis were induced. The combined treatment, however, did not potentiate either caspase-3 activity or apoptosis. Similar to previous findings we observed that Pc 4-PDT initiated a time-dependent extracellular TNF accumulation. The data suggest that: a) PDT and rhTNF induce cancer cell killing through different mechanisms; and b) Pc 4-PDT-induced TNF production is a stress response that may not directly affect photocytotoxicity.

  13. Fumonisin B1 does not prevent apoptosis in A431 human epidermoid carcinoma cells after photosensitization with a silicon phthalocyanine.

    PubMed

    Nagy, B; Chiu, S M; Separovic, D

    2000-09-01

    Photodynamic therapy with the phthalocyanine photosensitizer Pc 4 (Pc 4-PDT), an apoptosis inducer, is associated with accumulation of ceramide in various cell lines. The role of ceramide in Pc 4-PDT-induced apoptosis was investigated in A431 cells. Caspase-3 (casp-3) was activated and TUNEL positive cells began to appear 30 and 60 min post-Pc 4-PDT, respectively. A rapid increase (10 min) in cellular ceramide levels was observed after Pc 4-PDT. Induced ceramide accumulation was maintained over 60 min, Acid sphingomyelinase, a ceramide-generating enzyme, was inhibited after photosensitization with Pc 4, suggesting that the enzyme was not required for stimulated ceramide accumulation. Co-treatment of A431 cells with fumonisin B1, a ceramide synthase inhibitor, and Pc 4-PDT led to a decrease in ceramide levels without any effect on induced casp-3 activity or apoptosis. In the presence of zVAD, a pan-caspase inhibitor, apoptosis was abolished, while ceramide levels remained elevated after Pc 4-PDT. Exposure of A431 cells to exogenous C6-ceramide for 22 h, led to induction of apoptosis, and the process was abrogated by zVAD. In conclusion, C6-ceramide-, like Pc 4-PDT-induced apoptosis, is zVAD-sensitive. Furthermore, Pc 4 photosensitization can lead to apoptosis without FB-sensitive elevation in ceramide levels upstream of caspases.

  14. Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells.

    PubMed

    Chang, Chialun; Takayanagi, Atsushi; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2013-05-01

    Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

  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.

  16. Differential responses of skin cancer-chemopreventive agents silibinin, quercetin, and epigallocatechin 3-gallate on mitogenic signaling and cell cycle regulators in human epidermoid carcinoma A431 cells.

    PubMed

    Bhatia, N; Agarwal, C; Agarwal, R

    2001-01-01

    Silibinin, quercetin, and epigallocatechin 3-gallate (EGCG) have been shown to be skin cancer-preventive agents, albeit by several different mechanisms. Here, we assessed whether these agents show their cancer-preventive potential by a differential effect on mitogenic signaling molecules and cell cycle regulators. Treatment of human epidermoid carcinoma A431 cells with these agents inhibited the activation of the epidermal growth factor receptor and the downstream adapter protein Shc, but only silibinin showed a marked inhibition of mitogen-activated protein kinase-extracellular signal-regulated kinase-1 and -2 activation. In terms of cell cycle regulators, silibinin treatment showed an induction of Cip1/p21 and Kip1/p27 together with a significant decrease in cyclin-dependent kinase (CDK)-4, CDK2, and cyclin D1. Quercetin treatment, however, resulted in a moderate increase in Cip1/p21 with no change in Kip1/p27 and a decrease in CDK4 and cyclin D1. EGCG treatment also led to an induction of Cip1/p21 but no change in Kip1/27, CDK2, and cyclin D1 and a decrease in CDK4 only at low doses. Treatment of cells with these agents resulted in a strong dose- and time-dependent cell growth inhibition. A high dose of silibinin and low and high doses of quercetin and EGCG also led to cell death by apoptosis, suggesting that a lack of their inhibitory effect on mitogen-activated protein kinase-extracellular signal-regulated kinase-1 and -2 activation possibly "turns on" an apoptotic cell death response associated with their cancer-preventive and anticarcinogenic effects. Together, these results suggest that silibinin, quercetin, and EGCG exert their cancer-preventive effects by differential responses on mitogenic signaling and cell cycle regulators.

  17. 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; Wang, Qiong; He, Hao; Zang, Linghe; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Xia, Mingyu; Ikejima, Takashi

    2013-03-08

    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), such 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.

  18. Corticotropin-releasing factor induces phosphorylation of phospholipase C-gamma at tyrosine residues via its receptor 2beta in human epidermoid A-431 cells.

    PubMed

    Kiang, J G; Ding, X Z; Gist, I D; Jones, R R; Tsokos, G C

    1998-12-18

    This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-gamma via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2beta. The levels of the CRF receptor type 2beta were not altered in cells treated with activators of protein kinase C, Ca2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-beta3, -gamma1, or -gamma2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-gamma1 and -gamma2 were phosphorylated. The increase in phospholipase C-gamma phosphorylation was concentration-dependent with an EC50 of 4.2+/-0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2beta that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2iminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated the phospholipase C-gamma phosphorylation. In summary

  19. Adherence of skin bacteria to human epithelial cells.

    PubMed Central

    Romero-Steiner, S; Witek, T; Balish, E

    1990-01-01

    Aerobic and anaerobic bacteria isolated from human axillae were tested for their capacity to adhere to buccal epithelial cells, immortalized human epithelial (HEp-2) cells, and undifferentiated and differentiated human epithelial cells. In general, both aerobic and anaerobic diphtheroids adhered better to differentiated human epithelial cells than to HEp-2 and undifferentiated human epithelial cells (P less than 0.05). Mannose, galactose, fucose, N-acetyl-D-glucosamine, and fibronectin were also assayed for their capacity to inhibit the adherence of diphtheroids to human epithelial cells. A great deal of variability was observed in the capacity of the latter compounds to inhibit the attachment of aerobic diphtheroids to undifferentiated and differentiated epithelial cells. Overall, mannose appeared to be best at inhibiting the adherence of the aerobic diphtheroids to undifferentiated human epithelial cells. Galactose, fucose, N-acetyl-D-glucosamine, and fibronectin showed a greater capacity to inhibit attachment of aerobic diphtheroids to differentiated than to undifferentiated human epithelial cells. The inhibition of adherence to differentiated human epithelial cells varied with the microorganism and the compound tested; however, the highest and most consistent inhibition of adherence (76.1 to 88.6%) was observed with a 5% solution of N-acetyl-D-glucosamine. The in vitro adherence and adherence inhibition assays presented here demonstrate that a number of adhesins and receptors are involved in the adherence of skin bacteria to human epithelial cells and receptors on human epithelial cells are apparently altered during differentiation. PMID:2298877

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

    PubMed

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

    1977-12-15

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

  1. Accelerated degradation of 160 kDa epidermal growth factor (EGF) receptor precursor by the tyrosine kinase inhibitor herbimycin A in the endoplasmic reticulum of A431 human epidermoid carcinoma cells.

    PubMed Central

    Murakami, Y; Mizuno, S; Uehara, Y

    1994-01-01

    The effect of herbimycin A on the biosynthesis of epidermal growth factor (EGF) receptor was examined in human epidermoid carcinoma A431 cells. Cells were pulse-labelled with [35S]methionine, and EGF receptor biosynthesis was quantified by immunoprecipitation using a monoclonal anti-(EGF receptor) antibody. In the presence of herbimycin A, an immature 160 kDa EGF receptor precursor accumulated in 1 h and disappeared completely in 4 h. Pulse-labelled 160 kDa receptor precursor in the absence of herbimycin A, however, was converted normally into a 170 kDa one by chase with herbimycin A. Herbimycin A affected neither the synthesis of the secreted form of EGF receptor devoid of cytoplasmic domain, nor that of the transferrin receptor in A431 cells. The herbimycin A-induced degradation of 160 kDa EGF receptor precursor was not inhibited by an inhibitor of lysosomal enzymes, NH4Cl. Endoglycosidase H digestion of the 160 kDa precursor converted it into the deglycosylated 130 kDa precursor peptide. These results suggested that herbimycin A selectively acted on the EGF receptor precursor during the synthesis of the 160 kDa form, probably on the cytoplasmic domain, to form an aberrant molecule which was subjected to rapid degradation in the endoplasmic reticulum. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8037692

  2. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2009-10-01

    Appendix……………………………………………………………………………… 11 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A method for... Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability...Abstracts Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turshvili, Sam Aparicio , Joanne Emerman and Connie Eaves, “Identification of Human Mammary

  3. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

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

    2013-03-01

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

  4. Glucocorticoid receptor in human respiratory epithelial cells.

    PubMed

    Pujolsa, Laura; Mullol, Joaquim; Picado, Cèsar

    2009-01-01

    Inhaled and intranasal glucocorticoids (GCs) are the most common and effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, and asthma, and the respiratory epithelium is a primary target of GC anti-inflammatory actions. GC effects are mediated through the GC receptor (GR). In humans, one single GR gene gives rise to two main GR products, namely GRalpha and GRbeta, which are subject to translational and posttranslational modifications. GRalpha is expressed in virtually all human cells and tissues, including respiratory epithelial cells, and - at least in vitro - is downregulated by GC. GRalpha mediates the anti-inflammatory actions of GC by activating transcription of anti-inflammatory genes through binding of GRalpha to glucocorticoid response elements (GRE) located in the promoter region of target genes, repressing transcription of proinflammatory genes through direct interaction between GRalpha and proinflammatory transcription factors, such as AP-1 and NF-kappaB (transrepression), and also by destabilizing the mRNA of proinflammatory mediators. GRbeta acts as a dominant negative inhibitor of GRalpha-mediated transactivation and transrepression in certain in vitro studies with transfected cells. The GRbeta message is expressed at low levels in numerous tissues and its protein is mainly expressed in inflammatory cells, although it has also been detected in airway epithelial cells. Increased GRbeta expression has been reported in bronchial asthma and nasal polyposis, and after incubation of cells with certain proinflammatory stimuli. However, the role of GRbeta in modulating GC sensitivity in vivo has been highly debated and is as yet unclear.

  5. Focal epithelial hyperplasia caused by human papillomavirus 13.

    PubMed

    Saunders, Natasha R; Scolnik, Dennis; Rebbapragada, Anuradha; Koelink, Eric; Craw, Lindsey; Roth, Sherryn; Aronson, Leya; Perusini, Stephen; Silverman, Michael S

    2010-06-01

    Focal epithelial hyperplasia is a benign, papulo-nodular disease of the oral cavity. It is rare, affecting primarily Native American populations during childhood. It is closely associated with human papillomavirus 13 and 32. This report describes the diagnosis of 2 cases of focal epithelial hyperplasia in children from southern Guyana. The diagnosis was made using clinical criteria, polymerase chain reaction, and DNA sequencing.

  6. 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

  7. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells.

    PubMed

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-07-15

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.

  8. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells

    PubMed Central

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-01-01

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite. PMID:25221599

  9. Purification and properties of the alpha-3/4-L-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line.

    PubMed

    Johnson, P H; Donald, A S; Watkins, W M

    1993-04-01

    A soluble alpha-3/4-fucosyltransferase secreted into the growth medium of the human A431 epidermoid carcinoma cell line has been purified 700,000 fold by a series of steps involving chromatography on Phenyl Sepharose 4B, CM-Sephadex C-50 and GDP-hexanolamine Sepharose 4B. The untreated spent culture medium transferred almost ten times more fucose to the subterminal N-acetylglucosamine residue in the Type 1 (Gal beta 1-3GlcNAc) disaccharide than to the subterminal sugar in the Type 2 (Gal beta 1-4GlcNAc) disaccharide; the relative activity with these two substrates remained virtually unchanged throughout the purification procedure. At no stage was any alpha-3-fucosyltransferase species acting solely on N-acetylglucosamine residues in Type 2 chains separated from the bulk of the alpha-3/4-fucosyltransferase activity. The purified enzyme preparation showed insignificant activity with glycoprotein substrates having N-linked oligosaccharide chains with terminal Type 2 sequences but transferred fucose to a mucin-type glycoprotein with O-linked oligosaccharide chains with terminal Type 1 structures. Lactose was a poor substrate but the activity of the enzyme was influenced by the presence of substituents on the terminal beta-galactosyl residue and 2'-fucosyllactose was almost as good an acceptor as the Type 1 disaccharide. The properties of the purified enzyme with regard to specificity, divalent cation requirements, pH optimum, and M(r), closely resembled those of the Lewis-blood-group gene associated alpha-3/4-fucosyltransferase isolated from human milk.

  10. 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.

  11. 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.

  12. Development of an ErbB-overexpressing A-431 Optical Reporting Tumor Xenograft Model to Assess Targeted Photodynamic Therapy Regimens

    PubMed Central

    Savellano, Mark D.; Owusu-Brackett, Nicci; Son, Ji; Callier, Thierri; Savellano, Dagmar Högemann

    2010-01-01

    To better assess the efficacy of erbB-targeted therapies, it would help to have optical reporting human tumor xenograft models that abundantly express erbB receptors. A-431 cells have frequently been used in erbB1-targeting studies, but a well-characterized optical reporting version of the cell line has not been readily available. In this study, optical reporting A-431 clones were developed that express both a fluorescent protein reporter (green, GFP; or red, RFP) and a bioluminescent reporter, firefly luciferase. Reporter genes were transduced into cells using commercial lentiviral vectors, and clonal selection was carried out using a series of procedures. A number of clones were isolated for further characterization. A GFP/luciferase clone, A-431/D4, and an RFP/luciferase clone, A-431/G4, were obtained that exhibit erbB1 expression levels and tumor growth kinetics similar to the parental cells. To demonstrate the utility of the optical reporting clones, A-431/G4 tumors were grown subcutaneously in nude mice and treated with vascular-targeted photodynamic therapy (PDT), which targets the angiogenic consequences of erbB signaling. The A-431/G4 tumor model permitted highly sensitive longitudinal monitoring of PDT treatment response using optical imaging. A-431/D4 and A-431/G4 optical reporting tumor models should also prove useful for assessing therapies that directly target the erbB1 receptor. PMID:20880229

  13. 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

  14. 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.

  15. Bacillus anthracis lethal toxin reduces human alveolar epithelial barrier function.

    PubMed

    Langer, Marybeth; Duggan, Elizabeth Stewart; Booth, John Leland; Patel, Vineet Indrajit; Zander, Ryan A; Silasi-Mansat, Robert; Ramani, Vijay; Veres, Tibor Zoltan; Prenzler, Frauke; Sewald, Katherina; Williams, Daniel M; Coggeshall, Kenneth Mark; Awasthi, Shanjana; Lupu, Florea; Burian, Dennis; Ballard, Jimmy Dale; Braun, Armin; Metcalf, Jordan Patrick

    2012-12-01

    The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.

  16. Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function

    PubMed Central

    Langer, Marybeth; Duggan, Elizabeth Stewart; Booth, John Leland; Patel, Vineet Indrajit; Zander, Ryan A.; Silasi-Mansat, Robert; Ramani, Vijay; Veres, Tibor Zoltan; Prenzler, Frauke; Sewald, Katherina; Williams, Daniel M.; Coggeshall, Kenneth Mark; Awasthi, Shanjana; Lupu, Florea; Burian, Dennis; Ballard, Jimmy Dale; Braun, Armin

    2012-01-01

    The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness. PMID:23027535

  17. Zinc reduces epithelial barrier compromise induced by human seminal plasma

    PubMed Central

    Mullin, James M.; Diguilio, Katherine M.; Valenzano, Mary C.; Deis, Rachael; Thomas, Sunil; Zurbach, E. Peter; Abdulhaqq, Shaheed; Montaner, Luis J.

    2017-01-01

    Human semen has the potential to modulate the epithelial mucosal tissues it contacts, as seminal plasma (SP) is recognized to contain both pro- and anti-barrier components, yet its effects on epithelial barrier function are largely unknown. We addressed the role of human SP when exposed to the basal-lateral epithelial surface, a situation that would occur clinically with prior mechanical or disease-related injury of the human epithelial mucosal cell layers in contact with semen. The action of SP on claudins-2, -4, -5, and -7 expression, as well as on a target epithelium whose basolateral surface has been made accessible to SP, showed upregulation of claudins-4 and -5 in CACO-2 human epithelial cell layers, despite broad variance in SP-induced modulation of transepithelial electrical resistance and mannitol permeability. Upregulation of claudin-2 by SP also exhibited such variance by SP sample. We characterize individual effects on CACO-2 barrier function of nine factors known to be present abundantly in seminal plasma (zinc, EGF, citrate, spermine, fructose, urea, TGF, histone, inflammatory cytokines) to establish that zinc, spermine and fructose had significant potential to raise CACO-2 transepithelial resistance, whereas inflammatory cytokines and EGF decreased this measure of barrier function. The role of zinc as a dominant factor in determining higher levels of transepithelial resistance and lower levels of paracellular leak were confirmed by zinc chelation and exogenous zinc addition. As expected, SP presentation to the basolateral cell surface also caused a very dramatic yet transient elevation of pErk levels. Results suggest that increased zinc content in SP can compete against the barrier-compromising effect of negative modulators in SP when SP gains access to that epithelium’s basolateral surface. Prophylactic elevation of zinc in an epithelial cell layer prior to contact by SP may help to protect an epithelial barrier from invasion by SP-containing STD

  18. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2010-10-01

    breast is highly expressed by luminal epithelial cells and is less expressed by basal cells19,20. In contrast, CD49f (a6 integrin) has an inverse pattern...mouse stretched on its back. The hose and nose cone from the anesthetic vaporizer are securely attached to one side of the plate, and a heated pad is...the mouse by a nose cone. Check that the mouse has reached surgical anesthesia by loss of pedal withdrawal reflex . ! cautIon Institutional review

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

    SciTech Connect

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

    1999-02-01

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

  20. Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

    DTIC Science & Technology

    2005-07-01

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

  1. 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...

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

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

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

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

    DTIC Science & Technology

    2012-04-01

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

  4. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

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

    2007-11-10

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

  5. Human thymus contains amnion epithelial antigens.

    PubMed Central

    Hsi, B L; Yeh, C J; Faulk, W P

    1983-01-01

    Antibodies produced in rabbits to detergent-solubilized human amnion were found to react with Hassall's corpuscles in human thymus. Following nomenclature for placental antigens, the immunogenic group responsible for these antibodies has been tentatively designated as amnion antigens 1 (AA1). The anti-AA1 antisera did not react with other thymic components, nor did they react with any other extra-embryonic tissues than amniotic epithelium. Some adult ectodermally derived tissues, such as breast ductal and corneal epithelium, reacted with anti-AA1, but others such as skin and vagina did not. These findings link an antigenic relationship between amniotic epithelium and certain ectodermal derivatives. Amnion exists long before these tissues are formed, raising the possibility that amniotic epithelium may play an inductive role in their development. Images Figure 1 Figure 2 PMID:6343232

  6. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2007-10-01

    robust and reproducible methodology to detect, quantify and isolate stem cells in normal human mammary tissue, using a xenotransplantation system...covers the first year of the grant, during which substantial progress has been made in the development and validation of the xenotransplantation assay...Subrenal xenotransplantation surgery. The hair on the back of anesthetized mice was shaved, and the skin swabbed with 70% alcohol. An anterior to

  7. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2008-10-01

    9 Appendix……………………………………………………………………………… 10 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A...Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turashvili, Samuel Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human...Eirew, Afshin Raouf, John Stingl, Gulisa Turashvili, Allen Delaney, Joanne Emerman, Marco Marra and Samuel Aparicio . “Stem Cells in the Mammary Gland

  8. 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.

  9. Conserved two-step regulatory mechanism of human epithelial differentiation.

    PubMed

    Rane, Jayant K; Droop, Alastair P; Pellacani, Davide; Polson, Euan S; Simms, Matthew S; Collins, Anne T; Caves, Leo S D; Maitland, Norman J

    2014-02-11

    Human epithelia are organized in a hierarchical structure, where stem cells generate terminally differentiated cells via intermediate progenitors. This two-step differentiation process is conserved in all tissues, but it is not known whether a common gene set contributes to its regulation. Here, we show that retinoic acid (RA) regulates early human prostate epithelial differentiation by activating a tightly coexpressed set of 80 genes (e.g., TMPRSS2). Response kinetics suggested that some of these genes could be direct RA targets, whereas others are probably responding indirectly to RA stimulation. Comparative bioinformatic analyses of published tissue-specific microarrays and a large-scale transcriptomic data set revealed that these 80 genes are not only RA responsive but also significantly coexpressed in many human cell systems. The same gene set preferentially responds to androgens during terminal prostate epithelial differentiation, implying a cell-type-dependent interplay between RA and tissue-specific transcription factor-mediated signaling in regulating the two steps of epithelial differentiation.

  10. Hypoxia Increases Epithelial Permeability in Human Nasal Epithelia

    PubMed Central

    Min, Hyun Jin; Kim, Tae Hoon; Yoon, Joo-Heon

    2015-01-01

    Purpose The nasal mucosa is the first site to encounter pathogens, and it forms continuous barriers to various stimuli. This barrier function is very important in the innate defense mechanism. Additionally, inflammation of the nasal sinus is known to be a hypoxic condition. Here, we studied the effect of hypoxia on barrier function in normal human nasal epithelial (NHNE) cells. Materials and Methods The expression levels of various junction complex proteins were assessed in hypoxia-stimulated NHNE cells and human nasal mucosal tissues. We performed real-time polymerase chain reaction analysis, western blotting, and immunofluorescence assays to examine differences in the mRNA and protein expression of ZO-1, a tight junction protein, and E-cadherin in NHNE cells. Moreover, we evaluated the trans-epithelial resistance (TER) of NHNE cells under hypoxic conditions to check for changes in permeability. The expression of ZO-1 and E-cadherin was measured in human nasal mucosa samples by western blotting. Results Hypoxia time-dependently decreased the expression of ZO-1 and E-cadherin at the gene and protein levels. In addition, hypoxia decreased the TER of NHNE cells, which indicates increased permeability. Human nasal mucosa samples, which are supposed to be hypoxic, showed significantly decreased levels of ZO-1 and E-cadherin expression compared with control. Conclusion Our results demonstrate that hypoxia altered the expression of junction complex molecules and increased epithelial permeability in human nasal epithelia. This suggests that hypoxia causes barrier dysfunction. Furthermore, it may be associated with innate immune dysfunction after encountering pathogens. PMID:25837192

  11. 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.

  12. Expression of inducible nitric oxide in human lung epithelial cells.

    PubMed

    Robbins, R A; Barnes, P J; Springall, D R; Warren, J B; Kwon, O J; Buttery, L D; Wilson, A J; Geller, D A; Polak, J M

    1994-08-30

    Nitric oxide (NO) is increased in the exhaled air of subjects with several airway disorders. To determine if cytokines could stimulate epithelial cells accounting for the increased NO, the capacity of the proinflammatory cytokines (cytomix: tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma) to increase inducible nitric oxide synthase (iNOS) was investigated in A549 and primary cultures of human bronchial epithelial cells. Cytomix induced a time-dependent increase in nitrite levels in culture supernatant fluids (p < 0.05). Increased numbers of cells stained for iNOS and increased iNOS mRNA was detected in the cytokine-stimulated cells compared to control (p < 0.05). Dexamethasone diminished the cytokine-induced increase in nitrite, iNOS by immunocytochemistry, and iNOS mRNA. These data demonstrate that cytokines, such as those released by mononuclear cells, can induce lung epithelial iNOS expression and NO release, and that this is attenuated by dexamethasone.

  13. Asbestos exposure increases human bronchial epithelial cell fibrinolytic activity.

    PubMed

    Gross, T J; Cobb, S M; Gruenert, D C; Peterson, M W

    1993-03-01

    Chronic exposure to asbestos fibers results in fibrotic lung disease. The distal pulmonary epithelium is an early target of asbestos-mediated injury. Local plasmin activity may be important in modulating endoluminal inflammatory responses in the lung. We studied the effects of asbestos exposure on cell-mediated plasma clot lysis as a marker of pericellular plasminogen activation. Exposing human bronchial epithelial (HBE) cells to 100 micrograms/ml of asbestos fibers for 24 h resulted in increased plasma clot lysis. Fibrinolytic activity was augmented in a dose-dependent fashion, was not due to secreted protease, and occurred only when there was direct contact between the plasma clot and the epithelial monolayer. Further analysis showed that asbestos exposure increased HBE cell-associated urokinase-type plasminogen activator (uPA) activity in a time-dependent manner. The increased cell-associated PA activity could be removed by acid washing. The increase in PA activity following asbestos exposure required new protein synthesis because it was abrogated by treatment with either cycloheximide or actinomycin D. Therefore, asbestos exposure increases epithelial-mediated fibrinolysis by augmenting expression of uPA activity at the cell surface by mechanisms that require new RNA and protein synthesis. These observations suggest a novel mechanism whereby exposure of the distal epithelium to inhaled particulates may result in a chronic inflammatory response that culminates in the development of fibrotic lung disease.

  14. Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

    PubMed Central

    1995-01-01

    Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells. PMID:7559780

  15. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  16. Norepinephrine potentiates proinflammatory responses of human vaginal epithelial cells.

    PubMed

    Brosnahan, Amanda J; Vulchanova, Lucy; Witta, Samantha R; Dai, Yuying; Jones, Bryan J; Brown, David R

    2013-06-15

    The vaginal epithelium provides a barrier to pathogens and recruits immune defenses through the secretion of cytokines and chemokines. Several studies have shown that mucosal sites are innervated by norepinephrine-containing nerve fibers. Here we report that norepinephrine potentiates the proinflammatory response of human vaginal epithelial cells to products produced by Staphylococcus aureus, a pathogen that causes menstrual toxic shock syndrome. The cells exhibit immunoreactivity for catecholamine synthesis enzymes and the norepinephrine transporter. Moreover, the cells secrete norepinephrine and dopamine at low concentrations. These results indicate that norepinephrine may serve as an autocrine modulator of proinflammatory responses in the vaginal epithelium.

  17. Focal epithelial hyperplasia: a multifocal oral human papillomavirus infection.

    PubMed

    Flaitz, C M

    2000-01-01

    Widespread, slightly elevated and confluent nodules are observed throughout the oral mucosa in a young Hispanic girl. Repeated irritation of the soft tissues from a compromised occlusion is an aggravating factor for the spread of these lesions. A diagnosis of focal epithelial hyperplasia, a human papillomavirus infection, is made following histopathologic diagnosis and viral typing. Recognition of this specific type of warts is important in order to avoid the mistaken identification of condyloma acuminata, which may have significant repercussions in the life of a young child.

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

    SciTech Connect

    Twite, Nicolas; Andrei, Graciela; Kummert, Caroline; Donner, Catherine; Perez-Morga, David; De Vos, Rita; Snoeck, Robert; Marchant, Arnaud

    2014-07-15

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

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

    PubMed

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

    2014-04-01

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

  20. 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.

  1. Efficient cultivation conditions for human limbal epithelial cells.

    PubMed

    Kim, Mee Kum; Lee, Jae Lim; Oh, Joo Youn; Shin, Mi Sun; Shin, Kyeong Seon; Wee, Won Ryang; Lee, Jin Hak; Park, Ki Sook; Son, Young Sook

    2008-10-01

    To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

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

    PubMed

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

  4. 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.

  5. Tungsten-induced carcinogenesis in human bronchial epithelial cells

    PubMed Central

    Laulicht, Freda; Brocato, Jason; Cartularo, Laura; Vaughan, Joshua; Wu, Feng; Kluz, Thomas; Sun, Hong; Oksuz, Betul Akgol; Shen, Steven; Paena, Massimilano; Medici, Serenella; Zoroddu, Maria Antonietta; Costa, Max

    2015-01-01

    Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungsten’s ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer related pathways in transformed clones as determined by RNA seq. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data shows the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans. PMID:26164860

  6. Culture models of human mammary epithelial cell transformation

    SciTech Connect

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

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

  7. 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...

  8. Cytotoxicity of folpet fungicide on human bronchial epithelial cells.

    PubMed

    Canal-Raffin, Mireille; l'Azou, Béatrice; Jorly, Joana; Hurtier, Annabelle; Cambar, Jean; Brochard, Patrick

    2008-07-30

    Folpet, a widely used dicarboximide fungicide, has been detected in the ambient air of several vine-growing regions of France. It is present in particle form in the environment; however, no study exploring its potential health impact on airways and the respiratory system has been published. Here, the biological effect of these particles was investigated in vitro on human bronchial epithelial cells (16HBE14o-). To be close to the real-life conditions of exposure, Folpan 80WG, a commercial form of folpet, was tested. Folpan 80WG particles showed dose- and time-dependent cytotoxic effects on 16HBE14o- cells. This effect was compared to that produced by technical-grade folpet and both were found to induce a toxicity with similar IC(50) values after 24h of exposure. After 4h and at least until 48h of exposure, the IC(50) values of Folpan 80WG particles were between 2.4 and 2.8 microg/cm(2). Investigation of the cytotoxicity found that Folpan 80WG particles at 1.85 microg/cm(2) induced an increase in ROS production from the first hour of exposure. Evidence that oxidative processes occur in folpet-exposed cells was confirmed by the presence of membrane lipid peroxidation. Furthermore, early apoptosis and late apoptosis/necrosis were both present after the first hour of exposure. These findings indicate that exposure to Folpan 80WG particles result in a rapid cytotoxic effect on human bronchial epithelial cells in vitro that could be in part explained by oxidative stress, characterised by membrane lipid peroxidation and ROS production.

  9. Investigating the Responses of Human Epithelial Cells to Predatory Bacteria

    PubMed Central

    Monnappa, Ajay K.; Bari, Wasimul; Choi, Seong Yeol; Mitchell, Robert J.

    2016-01-01

    One beguiling alternative to antibiotics for treating multi-drug resistant infections are Bdellovibrio-and-like-organisms (BALOs), predatory bacteria known to attack human pathogens. Consequently, in this study, the responses from four cell lines (three human and one mouse) were characterized during an exposure to different predatory bacteria, Bdellovibrio bacteriovorus HD100, Bacteriovorus BY1 and Bacteriovorax stolpii EB1. TNF-α levels were induced in Raw 264.7 mouse macrophage cultures with each predator, but paled in comparison to those obtained with E. coli. This was true even though the latter strain was added at an 11.1-fold lower concentration (p < 0.01). Likewise, E. coli led to a significant (54%) loss in the Raw 264.7 murine macrophage viability while the predatory strains had no impact. Tests with various epithelial cells, including NuLi-1 airway, Caco2, HT29 and T84 colorectal cells, gave similar results, with E. coli inducing IL-8 production. The viabilities of the NuLi-1 and Caco-2 cells were slightly reduced (8%) when exposed to the predators, while T84 viability remained steady. In no cases did the predatory bacteria induce actin rearrangement. These results clearly demonstrate the gentle natures of predatory bacteria and their impacts on human cells. PMID:27629536

  10. 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 TEC 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.

  11. 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.

  12. 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.

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

    PubMed Central

    2004-01-01

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

  14. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods.

    PubMed

    Gildea, John J; McGrath, Helen E; Van Sciver, Robert E; Wang, Dora Bigler; Felder, Robin A

    2013-01-01

    The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.

  15. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

    SciTech Connect

    Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R. )

    1990-01-01

    Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV.

  16. Thymic epithelial cell development and its dysfunction in human diseases.

    PubMed

    Sun, Lina; Li, Hongran; Luo, Haiying; Zhao, Yong

    2014-01-01

    Thymic epithelial cells (TECs) are the key components in thymic microenvironment for T cells development. TECs, composed of cortical and medullary TECs, are derived from a common bipotent progenitor and undergo a stepwise development controlled by multiple levels of signals to be functionally mature for supporting thymocyte development. Tumor necrosis factor receptor (TNFR) family members including the receptor activator for NF κ B (RANK), CD40, and lymphotoxin β receptor (LT β R) cooperatively control the thymic medullary microenvironment and self-tolerance establishment. In addition, fibroblast growth factors (FGFs), Wnt, and Notch signals are essential for establishment of functional thymic microenvironment. Transcription factors Foxn1 and autoimmune regulator (Aire) are powerful modulators of TEC development, differentiation, and self-tolerance. Dysfunction in thymic microenvironment including defects of TEC and thymocyte development would cause physiological disorders such as tumor, infectious diseases, and autoimmune diseases. In the present review, we will summarize our current understanding on TEC development and the underlying molecular signals pathways and the involvement of thymus dysfunction in human diseases.

  17. The Effect of Silica Nanoparticles on Human Corneal Epithelial Cells

    PubMed Central

    Park, Joo-Hee; Jeong, Hyejoong; Hong, Jinkee; Chang, Minwook; Kim, Martha; Chuck, Roy S.; Lee, Jimmy K.; Park, Choul-Yong

    2016-01-01

    Ocular drug delivery is an interesting field in current research. Silica nanoparticles (SiNPs) are promising drug carriers for ophthalmic drug delivery. However, little is known about the toxicity of SiNPs on ocular surface cells such as human corneal epithelial cells (HCECs). In this study, we evaluated the cytotoxicity induced by 50, 100 and 150 nm sizes of SiNPs on cultured HCECs for up to 48 hours. SiNPs were up-taken by HCECs inside cytoplasmic vacuoles. Cellular reactive oxygen species generation was mildly elevated, dose dependently, with SiNPs, but no significant decrease of cellular viability was observed up to concentrations of 100 μg/ml for three different sized SiNPs. Western blot assays revealed that both cellular autophagy and mammalian target of rapamycin (mTOR) pathways were activated with the addition of SiNPs. Our findings suggested that 50, 100 and 150 nm sized SiNPs did not induce significant cytotoxicity in cultured HCECs. PMID:27876873

  18. 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.

  19. Carbocisteine inhibits rhinovirus infection in human tracheal epithelial cells.

    PubMed

    Yasuda, H; Yamaya, M; Sasaki, T; Inoue, D; Nakayama, K; Yamada, M; Asada, M; Yoshida, M; Suzuki, T; Nishimura, H; Sasaki, H

    2006-07-01

    The aim of the study was to examine the effects of a mucolytic drug, carbocisteine, on rhinovirus (RV) infection in the airways. Human tracheal epithelial cells were infected with a major-group RV, RV14. RV14 infection increased virus titres and the cytokine content of supernatants. Carbocisteine reduced supernatant virus titres, the amount of RV14 RNA in cells, cell susceptibility to RV infection and supernatant cytokine concentrations, including interleukin (IL)-6 and IL-8, after RV14 infection. Carbocisteine reduced the expression of mRNA encoding intercellular adhesion molecule (ICAM)-1, the receptor for the major group of RVs. It also reduced the supernatant concentration of a soluble form of ICAM-1, the number and fluorescence intensity of acidic endosomes in the cells before RV infection, and nuclear factor-kappaB activation by RV14. Carbocisteine also reduced the supernatant virus titres of the minor group RV, RV2, although carbocisteine did not reduce the expression of mRNA encoding a low density lipoprotein receptor, the receptor for RV2. These results suggest that carbocisteine inhibits rhinovirus 2 infection by blocking rhinovirus RNA entry into the endosomes, and inhibits rhinovirus 14 infection by the same mechanism as well as by reducing intercellular adhesion molecule-1 levels. Carbocisteine may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection.

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

    PubMed

    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.

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

    PubMed Central

    Birch, Nigel P.; Suresh, Vinod

    2016-01-01

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

  2. Binding of transcobalamin II by human mammary epithelial cells.

    PubMed

    Adkins, Y; Lönnerdal, B

    2001-01-01

    The presence of nutrient binders in milk may have an important role during milk production and may influence the nutrient's bioavailability to the infant. Human milk and plasma contain at least two types of vitamin B12 binders: transcobalamin II (TCII) and haptocorrin (Hc). Vitamin B12 in milk is exclusively bound to Hc (Hc-B12). In plasma, the major vitamin B12 binding protein that is responsible for delivering absorbed vitamin B12 to most tissues and cells is TCII (TCII-B12). Currently, little is known about the route of secretion of vitamin B12 into human milk. It is possible that a receptor-mediated pathway is involved, since maternal vitamin B12 supplementation increases the amount of the vitamin secreted into human milk if the mother's vitamin B12 consumption is low, but remains unchanged if her intake is adequate. In this study, we investigated the process by which the mammary gland acquires vitamin B12 from maternal circulation, whether as a free vitamin or as a Hc-B12 or TCII-B12 complex. TCII was purified from plasma incubated with [57Co]vit B12 (B12*), while Hc was purified from whey incubated with B12*. Both proteins were separated by fast protein liquid chromatography using gel filtration and anion-exchange columns. Purity of the separated proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding studies were carried out on a monolayer of normal human mammary epithelial cells (HMEC) at 4 degrees C using free B12* and TCII-B12* and Hc-B12* complexes. Minimal binding of free B12* and Hc-B12* to HMEC was observed; however, HMEC exhibited a high affinity for the TCII-B12* complex. This study suggests that a specific cell surface receptor for the TCII-B12 complex exists in the mammary gland. It is possible that once vitamin B12 is in the mammary gland it is transferred to Hc (which may be synthesized by the mammary gland) and then secreted into milk as a Hc-B12 complex.

  3. 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...

  4. 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

  5. Connexin expression in nonneoplastic human prostate epithelial cells.

    PubMed

    Saladino, Francesca; Carruba, Giuseppe; Quader, Salmaan T A; Amoroso, Maria; Di Cristina, Antoniette; Webber, Mukta M; Castagnetta, Luigi A M

    2002-06-01

    Expression of gap-junction proteins connexins (Cx), specifically Cx43, Cx32, and Cx26, in both nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells as well as in two cell clones (WPEI-7 and WPEI-10) originating from the RWPE-1 cell line was investigated. The aim was to determine whether individual connexins are differentially expressed in cultured cells. Western blot analysis revealed striking differences in the expression of individual connexins in the cell lines studied. In particular, Cx43 is largely expressed in RWPE-1 and WPEI-10 cells, whereas Cx32 is expressed predominantly in RWPE-2 and WPEI-7 cells. In addition, both forskolin and estrone increase Cx43 expression levels in WPEI-10 cells, with no apparent effect on WPEI-7 cells. Conversely, forskolin and especially estrone induce a marked increase of Cx32 in WPEI-7 cells, whereas Cx32 expression is limitedly affected by both agents in WPEI-10 cells. Overall, expression levels of Cx43 and Cx32 appear to be inversely related, with RWPE-1 and WPEI-10 cells having a significantly higher Cx43 to Cx32 ratio than that observed in RWPE-2 and WPEI-7 cells. We recently reported that junctional communication could be rescued in RWPE-1 cells by either forskolin or estrone and that restoration of GJIC is associated with an increase of Cx43 or a decrease of Cx32, or both, eventually leading to a marked rise of the Cx43 to Cx32 ratio. Studies are currently ongoing in our laboratories to assess the potential effect of agents increasing the Cx43 to Cx32 ratio on GJIC activity in these systems.

  6. Cytotoxic Effects of Curcumin in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Hollborn, Margrit; Chen, Rui; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas; Kohen, Leon

    2013-01-01

    Backround Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro. Methodology/Principal Findings Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. Conclusion It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of

  7. Strategies to enhance epithelial-mesenchymal interactions for human hair follicle bioengineering.

    PubMed

    Ohyama, Manabu; Veraitch, Ophelia

    2013-05-01

    Hair follicle morphogenesis and regeneration depend on intensive but well-orchestrated interactions between epithelial and mesenchymal components. Accordingly, the enhancement of this crosstalk represents a promising approach to achieve successful bioengineering of human hair follicles. The present article summarizes the techniques, both currently available and potentially feasible, to promote epithelial-mesenchymal interactions (EMIs) necessary for human hair follicle regeneration. The strategies include the preparation of epithelial components with high receptivity to trichogenic dermal signals and/or mesenchymal cell populations with potent hair inductive capacity. In this regard, bulge epithelial stem cells, keratinocytes predisposed to hair follicle fate or keratinocyte precursor cells with plasticity may provide favorable epithelial cell populations. Dermal papilla cells sustaining intrinsic hair inductive capacity, putative dermal papilla precursor cells in the dermal sheath/neonatal dermis or trichogenic dermal cells derived from undifferentiated stem/progenitor cells are promising candidates as hair inductive dermal cells. The most established protocol for in vivo hair follicle reconstitution is co-grafting of epithelial and mesenchymal components into immunodeficient mice. In theory, combination of individually optimized cellular components of respective lineages should elicit most intensive EMIs to form hair follicles. Still, EMIs can be further ameliorated by the modulation of non-cell autonomous conditions, including cell compartmentalization to replicate the positional relationship in vivo and humanization of host environment by preparing human stromal bed. These approaches may not always synergistically intensify EMIs, however, step-by-step investigation probing optimal combinations should maximally enhance EMIs to achieve successful human hair follicle bioengineering.

  8. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells.

    PubMed

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W Michael; Das, Mita

    2014-04-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.

  9. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells

    PubMed Central

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W. Michael

    2014-01-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases. PMID:24500281

  10. Directed differentiation of airway epithelial cells of human bone marrow mesenchymal stem cells.

    PubMed

    Li, Jian-Dong

    2016-11-01

    The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

  11. The human thymus microenvironment: heterogeneity detected by monoclonal anti-epithelial cell antibodies.

    PubMed Central

    de Maagd, R A; MacKenzie, W A; Schuurman, H J; Ritter, M A; Price, K M; Broekhuizen, R; Kater, L

    1985-01-01

    Monoclonal antibodies were raised against human thymus stromal cells and their specificity for the epithelial component of thymus stroma assessed by double immunofluorescence using anti-keratin antibodies to identify epithelium. Our monoclonal antibodies identify six distinct patterns of epithelial cell antigen expression within the thymus: pan epithelial (antibody IP1); cortex (MR3 and MR6); cortical/medullary junction (IP2); subcapsule and subpopulation of medulla (MR10/MR14); Hassall's corpuscles and adjacent subpopulation of medulla (IP3); Hassall's corpuscles only (MR13/IP4). This heterogeneity of antigen expression suggests that many different epithelial microenvironments exist within the human thymus. Images Figure 1 Figure 1 Cont Figure 2 PMID:3884494

  12. [Focal epithelial hyperplasia of the oral mucosa. A unique manifestation of human papillomavirus].

    PubMed

    van der Voort, E A M; Arani, S Fallah; Hegt, V Noordhoek; van Praag, M C G

    2009-03-01

    A 34-year old Creole woman appeared at the dermatology department with white-pink spots on the oral mucosa, which had been there for some time. Histology showed lesions characteristic of focal epithelial hyperplasia. The patient was treated with a CO2 laser. Focal epithelial hyperplasia is a rare benign lesion and is caused by human papillomavirus subtypes 13 or 32; it only appears on the oral mucosa.

  13. Nicotine-induced epithelial-mesenchymal transition via Wnt/β-catenin signaling in human airway epithelial cells.

    PubMed

    Zou, Weifeng; Zou, Yimin; Zhao, Zhuxiang; Li, Bing; Ran, Pixin

    2013-02-15

    Epithelial-mesenchymal transition (EMT) has been proposed to be a mechanism in airway remodeling, which is a characteristic of chronic obstructive pulmonary disease (COPD). Studies have shown that cigarette smoke and nicotine are factors that induce Wnt/β-catenin activation, which is a pathway that has also been implicated in EMT. The main aim of this study was to test whether human bronchial epithelial cells are able to undergo EMT in vitro following nicotine stimulation via the Wnt3a/β-catenin signaling pathway. We show that nicotine activates the Wnt3a signal pathway, which leads to the translocation of β-catenin into the nucleus and activation of β-catenin/Tcf-dependent transcription in the human bronchial epithelial cell (HBEC) line. This accumulation was accompanied by an increase in smooth muscle actin, vimentin, matrix metalloproteinases-9, and type I collagen expression as well as downregulation of E-cadherin, which are typical characteristics of EMT. We also noted that the release of TGF-β(1) from these cells was stimulated by nicotine. Knockdown of Wnt3a with small interfering RNA (siRNA) prevented these effects, implying that β-catenin activation in these responses is Wnt3a dependent. Furthermore, specific knockdown of TGF-β(1) with TGF-β(1) siRNA partially prevented nicotine-induced EMT, suggesting that TGF-β(1) has a role in nicotine-mediated EMT in HBECs. These results suggest that HBECs are able to undergo EMT in vitro upon nicotine stimulation via the Wnt3a/β-catenin signaling pathway.

  14. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung epithelial cells.

    PubMed

    Xie, Hong; Smith, Leah J; Holmes, Amie L; Zheng, Tongzhang; Pierce Wise, John

    2016-05-01

    Cobalt is a toxic metal used in various industrial applications leading to adverse lung effects by inhalation. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells, especially normal lung epithelial cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in normal primary human lung epithelial cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble and particulate cobalt induced similar cytotoxicity while soluble cobalt was more genotoxic than particulate cobalt. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung epithelial cells.

  15. Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18.

    PubMed

    Bello, D; Webber, M M; Kleinman, H K; Wartinger, D D; Rhim, J S

    1997-06-01

    Prostate cancer and benign tumors of the prostate are the two most common neoplastic diseases in men in the United States, however, research on their causes and treatment has been slow because of the difficulty in obtaining fresh samples of human tissue and a lack of well characterized cell lines which exhibit growth and differentiation characteristics of normal prostatic epithelium. Non-neoplastic adult human prostatic epithelial cells from a white male donor were immortalized with human papillomavirus 18 which resulted in the establishment of the RWPE-1 cell line. Cells from the RWPE-1 cell line were further transformed by v-Ki-ras to establish the RWPE-2 cell line. The objectives of this study were to: (1) establish the prostatic epithelial origin and androgen responsiveness of RWPE-1 and RWPE-2 cell lines; (2) examine their response to growth factors; and (3) establish the malignant characteristics of the RWPE-2 cell line. Immunoperoxidase staining showed that both RWPE-1 and RWPE-2 cells express cytokeratins 8 and 18, which are characteristic of luminal prostatic epithelial cells, but they also coexpress basal cell cytokeratins. These cell lines show growth stimulation and prostate specific antigen (PSA) and androgen receptor (AR) expression in response to the synthetic androgen mibolerone, which establishes their prostatic epithelial origin. Both cell lines also show a dose-dependent growth stimulation by EGF and bFGF and growth inhibition when exposed to TGF-beta, however, the transformed RWPE-2 cells are less responsive. RWPE-1 cells neither grow in agar nor form tumors when injected into nude mice with or without Matrigel. However, RWPE-2 cells form colonies in agar and tumors in nude mice. In the in vitro invasion assay, RWPE-1 cells are not invasive whereas RWPE-2 cells are invasive. Nuclear expression of p53 and Rb proteins was heterogeneous but detectable by immunostaining in both cell lines. The RWPE-1 cells, which show many normal cell

  16. S1P differentially regulates migration of human ovarian cancer and human ovarian surface epithelial cells

    PubMed Central

    Wang, Dongmei; Zhao, Zhenwen; Caperell-Grant, Andrea; Yang, Gong; Mok, Samuel C.; Liu, Jinsong; Bigsby, Robert M.; Xu, Yan

    2009-01-01

    Epithelial ovarian cancer (EOC) arises from the epithelial layer covering the surface of ovaries and intra-peritoneal metastasis is commonly observed at diagnosis. Sphingosine-1-phosphate (S1P), a bioactive lipid signaling molecule, is potentially involved in EOC tumorigenesis. We have found that S1P is elevated in human EOC ascites. We show that physiologically relevant concentrations of S1P stimulate migration and invasion of EOC cells, but inhibit migration of human ovarian surface epithelial (HOSE) cells. In addition, S1P inhibits lysophosphatidic acid (LPA)-induced cell migration in HOSE, but not in EOC cells. We have provided the first line of evidence that the expression levels of S1P receptor subtypes are not the only determinants for how cells respond to S1P. Even though S1P1 is expressed and functional in HOSE cells, the inhibitory effect mediated by S1P2 is dominant in those cells. The cellular pre-existing stress fibers are also important determinants for the migratory response to S1P. Differential S1P-induced morphology changes are noted in EOC and HOSE cells. Pre-existing stress fibers in HOSE cells are further enhanced by S1P treatment, resulting in the negative migratory response to S1P. By contrast, EOC cells lost stress fibers and S1P treatment induces filopodium-like structures at cell edges, which correlates with increased cell motility. In addition, inhibition of the protein kinase C pathway is likely to be involved in the inhibitory effect of S1P on LPA-induced cell migration in HOSE cells. These findings are important for the development of new therapeutics targeting S1P and LPA in EOC. PMID:18645009

  17. 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...

  18. 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...

  19. 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...

  20. Human eccrine sweat gland epithelial cultures express ductal characteristics.

    PubMed Central

    Brayden, D J; Cuthbert, A W; Lee, C M

    1988-01-01

    1. Isolated human eccrine sweat glands were cultured in vitro. Cells were harvested and plated onto permeable supports to form confluent cell sheets, area 0.2 cm2. These were used to study the electrogenic transepithelial transport of ions by measurement of short-circuit current (SCC). Epithelial sheets had a basal SCC of 5.89 +/- 0.62 microA cm-2 (n = 33) and a transepithelial resistance of 74.1 +/- 5.6 omega cm2 (n = 33). The transepithelial potential difference varied between -0.2 and -1.8 mV with a mean value of -0.71 +/- 0.09 mV (n = 33). 2. The basal current was abolished by addition of 10 microM-amiloride to the apical bathing solution. The concentration of amiloride which inhibited basal SCC by 50% (EC50) was 0.4 microM. Cultures prepared from the secretory coil of sweat glands, rather than from whole glands, were similarly sensitive to amiloride (EC50 = 0.8 microM). 3. Lysylbradykinin (LBK), carbachol, isoprenaline, prostaglandin E2 (PGE2) and A23187 all increased SCC in cultures from whole glands. LBK responses were obtained with basolateral and not with apical application. Furthermore LBK actions were not substantially altered by cyclo-oxygenase inhibition but showed marked desensitization upon repeated application. Sheet cultures prepared from sweat gland coils also showed SCC responses to both carbachol and LBK. Forskolin, an activator of adenylate cyclase, did not alter SCC in either type of preparation. 4. Replacement of chloride and of chloride and bicarbonate in the bathing solution did not cause attenuation of the responses to LBK or carbachol in whole-gland sheet cultures. Furthermore responses were unaffected by piretanide or acetazolamide. These results were taken to indicate that anion secretion was not the basis for the SCC responses. 5. Responses to LBK and carbachol were significantly reduced by amiloride (10 microM), this effect being reversible. No responses to LBK or carbachol were seen when N-methyl-D-glucamine (NMDG) was used to

  1. The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

    SciTech Connect

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

    2001-05-12

    The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neoplastic breast epithelial cells may be substantially more plastic in their differentiation repertoire than previously anticipated. Thus, along with an increasing availability of markers for the myoepithelial lineage, at least a partial differentiation towards this lineage is being revealed frequently. It has also become clear that conversions towards the mesenchymal lineage actually occur, referred to as epithelial to mesenchymal transitions. Indeed, some of the so-called myofibroblasts surrounding the tumor may indeed have an epithelial origin rather than a mesenchymal origin. Because myoepithelial cells, epithelial to mesenchymal transition-derived cells, genuine stromal cells and myofibroblasts share common markers, we now need to define a more ambitious set of markers to distinguish these cell types in the microenvironment of the tumors. This is necessary because the different microenvironments may confer different clinical outcomes. The aim of this commentary is to describe some of the inherent complexities in defining cellular phenotypes in the microenvironment of breast cancer and to expand wherever possible on the implications for tumor suppression and progression.

  2. Tumorigenic transformation of human breast epithelial cells induced by mitochondrial DNA depletion.

    PubMed

    Kulawiec, Mariola; Safina, Alfiya; Desouki, Mohamed Mokhtar; Still, Ivan; Matsui, Sei-Ichi; Bakin, Andrei; Singh, Keshav K

    2008-11-01

    Human mitochondrial DNA (mtDNA) encodes 13 proteins involved in oxidative phosphorylation (OXPHOS). In order to investigate the role of mitochondrial OXPHOS genes in breast tumorigenesis, we have developed a breast epithelial cell line devoid of mtDNA (rho(0) cells). Our analysis revealed that depletion of mtDNA in breast epithelial cells results in in vitro tumorigenic phenotype as well as breast tumorigenesis in a xenograft model. We identified two major gene networks which were differentially regulated between parental and rho(0) epithelial cells. The focal proteins in these networks include (i) FN1 (fibronectin) and (ii) p53. Bioinformatic analyses of FN1 network identified laminin, integrin and 3 of 6 members of peroxiredoxin whose expression were altered in rho(0) epithelial cells. In the p53 network, we identified SMC4 and WRN whose changes in expression suggest that this network may affect chromosomal stability. Consistent with above finding our study revealed an increase in DNA double strand breaks and unique chromosomal rearrangements in rho(0) breast epithelial cells. Additionally, we identified tight junction proteins claudin-1 and claudin-7 in p53 network. To determine the functional relevance of altered gene expression, we focused on detailed analyses of claudin-1 and -7 proteins in breast tumorigenesis. Our study determined that (i) claudin-1 and 7 were indeed downregulated in rho(0) breast epithelial cells, (ii) downregulation of claudin-1 or -7 led to neoplastic transformation of breast epithelial cells, and (iii) claudin-1 and -7 were also downregulated in primary breast tumors. Together, our study suggest that mtDNA encoded OXPHOS genes play a key role in transformation of breast epithelial cells and that multiple pathway involved in mitochondria-to-nucleus retrograde regulation contribute to transformation of breast epithelial cells.

  3. 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.

  4. Epithelial expression and chromosomal location of human TLE genes: Implications for notch signaling and neoplasia

    SciTech Connect

    Liu, Yanling; Dehni, Ghassan; Stifani, S.

    1996-01-01

    The TLE genes are the human homologues of Drosophila groucho, a member of the Notch signaling pathway. This pathway controls a number of different cell-fate choices in invertebrates and vertebrates. We are interested in investigating the functions of the TLE gene family during epithelial determination and carcinogenesis. We show that expression of individual TLE genes correlates with immature epithelial cells that are progressing toward their terminally differentiated state, suggesting a role during epithelial differentiation. In both normal tissues and conditions resulting from incorrect or incomplete maturation events, such as metaplastic and neoplastic transformations, TLE expression is elevated and coincides with Notch expression, implicating these molecules in the maintenance of the undifferentiated state in epithelial cells. We also show that TLE1 and TLE2 are organized in a tandem array at chromosomal location 19p13.3, while TLE3 maps to 15q22. 26 refs., 4 figs.

  5. An improved method for isolation of epithelial and stromal cells from the human endometrium

    PubMed Central

    MASUDA, Ayako; KATOH, Noriko; NAKABAYASHI, Kazuhiko; KATO, Kiyoko; SONODA, Kenzo; KITADE, Mari; TAKEDA, Satoru; HATA, Kenichiro; TOMIKAWA, Junko

    2016-01-01

    We aimed to improve the efficiency of isolating endometrial epithelial and stromal cells (EMECs and EMSCs) from the human endometrium. We revealed by immunohistochemical staining that the large tissue fragments remaining after collagenase treatment, which are usually discarded after the first filtration in the conventional protocol, consisted of glandular epithelial and stromal cells. Therefore, we established protease treatment and cell suspension conditions to dissociate single cells from the tissue fragments and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. Four independent experiments showed that, on average, 1.2 × 106 of EMECs and 2.8 × 106 EMSCs were isolated from one hysterectomy specimen. We confirmed that the isolated cells presented transcriptomic features highly similar to those of epithelial and stromal cells obtained by the conventional method. Our improved protocol facilitates future studies to better understand the molecular mechanisms underlying the dynamic changes of the endometrium during the menstrual cycle. PMID:26853786

  6. 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

  7. Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche.

    PubMed

    Massie, Isobel; Dziasko, Marc; Kureshi, Alvena; Levis, Hannah J; Morgan, Louise; Neale, Michael; Sheth, Radhika; Tovell, Victoria E; Vernon, Amanda J; Funderburgh, James L; Daniels, Julie T

    2015-01-01

    The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein.

  8. Effects of titanium dioxide nanoparticles in human gastric epithelial cells in vitro.

    PubMed

    Botelho, Monica Catarina; Costa, Carla; Silva, Susana; Costa, Solange; Dhawan, Alok; Oliveira, Paula A; Teixeira, João P

    2014-02-01

    Manufacturing or using nanomaterials may result in exposure of workers to nanoparticles. Potential routes of exposure include skin, lung and gastrointestinal tract. The lack of health-based standards for nanomaterials combined with their increasing use in many different workplaces and products emphasize the need for a reliable temporary risk assessment tool. Therefore, the aim of this work was to explore the effects of different doses of titanium dioxide nanoparticles on human gastric epithelial cells in vitro. We analyzed proliferation by MTT assay, apoptosis by Tunel, migration by injury assay, oxidative stress by determining GSH/GSSG ratio and DNA damage by Comet assay on nanoparticle-treated AGS human gastric epithelial cell line in comparison to controls. We show and discuss the tumor-like phenotypes of nanoparticles-exposed AGS cells in vitro, as increased proliferation and decreased apoptosis. Our results demonstrate for the first time that nanoparticles induce tumor-like phenotypes in human gastric epithelial cells.

  9. Advanced Imaging and Tissue Engineering of the Human Limbal Epithelial Stem Cell Niche

    PubMed Central

    Massie, Isobel; Dziasko, Marc; Kureshi, Alvena; Levis, Hannah J.; Morgan, Louise; Neale, Michael; Sheth, Radhika; Tovell, Victoria E.; Vernon, Amanda J.; Funderburgh, James L.; Daniels, Julie T.

    2015-01-01

    The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein. PMID:25388395

  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. Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition.

    PubMed

    Andarawewa, Kumari L; Erickson, Anna C; Chou, William S; Costes, Sylvain V; Gascard, Philippe; Mott, Joni D; Bissell, Mina J; Barcellos-Hoff, Mary Helen

    2007-09-15

    Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  12. Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis.

    PubMed

    Malyukova, Irina; Murray, Karen F; Zhu, Chengru; Boedeker, Edgar; Kane, Anne; Patterson, Kathleen; Peterson, Jeffrey R; Donowitz, Mark; Kovbasnjuk, Olga

    2009-01-01

    Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.

  13. Differential growth factor induction and modulation of human gastric epithelial regeneration

    SciTech Connect

    Tetreault, Marie-Pier; Chailler, Pierre; Rivard, Nathalie; Menard, Daniel . E-mail: Daniel.Menard@USherbrooke.ca

    2005-05-15

    While several autocrine/paracrine growth factors (GFs) can all stimulate epithelial regeneration in experimentally wounded primary gastric cultures, clinical relevance for their non-redundant cooperative actions in human gastric ulcer healing is suggested by the sequential pattern of GF gene induction in vivo. Using new HGE cell lines able to form a coherent monolayer with tight junctions as well as using primary human gastric epithelial cultures, we show that EGF, TGF{alpha}, HGF and IGFs accelerate epithelial restitution upon wounding, independently of the TGF{beta} pathway (as opposed to intestinal cells). However, they differently modulate cell behavior: TGF{alpha} exerts strong effects (even more than EGF) on cytoplasmic spreading and non-oriented protruding activity of bordering cells whereas HGF preferentially coordinates single lamella formation, cell elongation and migration into the wound. IGF-I and IGF-II rather induce the alignment of bordering cells and maintain a compact monolayer front. The number of mitotic cells maximally increases with EGF, followed by TGF{alpha} and IGF-I,-II. The current study demonstrates that GFs differentially regulate the regeneration of human gastric epithelial cells through specific modulation of cell shape adaptation, migration and proliferation, further stressing that a coordination of GF activities would be necessary for the normal progression of post-wounding epithelial repair.

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

    PubMed

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

    2011-12-01

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

  15. Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16.

    PubMed Central

    Apt, D; Chong, T; Liu, Y; Bernard, H U

    1993-01-01

    The transcription of human papillomavirus type 16 (HPV-16) is mediated by the viral enhancer. Epithelial cell-specific activation is achieved by the cooperative interaction of apparently ubiquitous transcriptional factors. One of them, nuclear factor I (NFI), binds seven sites within the HPV-16 enhancer. Point mutations on enhancer fragments, which retain epithelial cell specificity, verify the functional contribution of NFI. In band shift experiments, the epithelial cell-derived NFI proteins CTF-1, CTF-2, and CTF-3 form a characteristic pattern of heterodimeric complexes which are observed in all epithelial cells tested. Divergence from this pattern in fibroblasts, liver cells, and lymphoid cells correlates with the lack of HPV-16 enhancer activation. The HPV-16 enhancer can be activated by CTF-1 in SL-2 cells, which lack NFI-like proteins. However, exogenous CTF-1 fails to overcome the inactivity of the viral enhancer in fibroblasts. Western immunoblot and supershift analysis shows that exogenously introduced CTF-1 proteins form different heterodimer complexes with the given subset of endogenous NFI proteins in epithelial or fibroblast cells. Polymerase chain reaction analysis and cDNA library screens identified the endogenous fibroblast type NFI as NFI-X, an NFI family member originally cloned from hamster liver cells. The strict correlation between the activation or lack of activation of the HPV-16 enhancer and cell-specific subsets of NFI proteins argues for the pivotal role of NFI binding sites in the epithelial cell-specific function of the viral enhancer. Images PMID:8392590

  16. Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling

    SciTech Connect

    Seomun, Young; Joo, Choun-Ki

    2008-07-18

    Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins {alpha}2 and {beta}1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin {beta}1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

  17. 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.

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

    SciTech Connect

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

    1990-11-01

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

  19. 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.

  20. Feasibility of a 3D human airway epithelial model to study respiratory absorption.

    PubMed

    Reus, Astrid A; Maas, Wilfred J M; Jansen, Harm T; Constant, Samuel; Staal, Yvonne C M; van Triel, Jos J; Kuper, C Frieke

    2014-03-01

    The respiratory route is an important portal for human exposure to a large variety of substances. Consequently, there is an urgent need for realistic in vitro strategies for evaluation of the absorption of airborne substances with regard to safety and efficacy assessment. The present study investigated feasibility of a 3D human airway epithelial model to study respiratory absorption, in particular to differentiate between low and high absorption of substances. Bronchial epithelial models (MucilAir™), cultured at the air-liquid interface, were exposed to eight radiolabeled model substances via the apical epithelial surface. Absorption was evaluated by measuring radioactivity in the apical compartment, the epithelial cells and the basolateral culture medium. Antipyrine, caffeine, naproxen and propranolol were highly transported across the epithelial cell layer (>5%), whereas atenolol, mannitol, PEG-400 and insulin were limitedly transported (<5%). Results indicate that the 3D human airway epithelial model used in this study is able to differentiate between substances with low and high absorption. The intra-experimental reproducibility of the results was considered adequate based on an average coefficient of variation (CV) of 15%. The inter-experimental reproducibility of highly absorbed compounds was in a similar range (CV of 15%), but this value was considerably higher for those compounds that were limitedly absorbed. No statistical significant differences between different donors and experiments were observed. The present study provides a simple method transposable in any lab, which can be used to rank the absorption of chemicals and pharmaceuticals, and is ready for further validation with respect to reproducibility and capacity of the method to predict respiratory transport in humans.

  1. 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.

  2. 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.

  3. Bio-synthesis of gold nanoparticles by human epithelial cells, in vivo.

    PubMed

    Larios-Rodriguez, E; Rangel-Ayon, C; Castillo, S J; Zavala, G; Herrera-Urbina, R

    2011-09-02

    Healthy epithelial cells, in vivo, have the ability to synthesize gold nanoparticles when aqueous tetrachloroauric acid is made to react with human skin. Neither a reducing agent nor a protecting chemical is needed for this bio-synthesis method. The first indication of gold nanoparticle formation is the staining of the skin, which turns deep purple. Stereoscopic optical micrographs of human skin tissue in contact with aqueous tetrachloroauric acid clearly show the staining of the epithelial cells. The UV-Vis spectrum of these epithelial cells shows an absorption band with a maximum at 553 nm. This absorption peak is within the wavelength region where the surface plasmon resonance (SPR) band of aqueous colloidal gold exhibits a maximum. Transmission electron micrographs show that gold nanoparticles synthesized by epithelial cells have sizes between 1 and 100 nm. The electron diffraction pattern of these nanoparticles reveals a crystalline structure whose interplanar distances correspond to fcc metallic gold. Transmission electron micrographs of ultra-thin (70 nm thick) slices of epithelial cells clearly and undoubtedly demonstrate that gold nanoparticles are inside the cell. According to high resolution transmission electron micrographs of intracellular single gold nanoparticles, they have the shape of a polyhedron.

  4. 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

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

    PubMed

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

    2015-07-16

    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.

  6. Influence of sex on gene expression in human corneal epithelial cells

    PubMed Central

    Suzuki, Tomo; Richards, Stephen M.; Liu, Shaohui; Jensen, Roderick V.

    2009-01-01

    Purpose Sex-associated differences have been identified in the anatomy, physiology and pathophysiology of the human cornea. We hypothesize that many of these differences are due to fundamental variations in gene expression. Our objective in this study was to determine whether such differences exist in human corneal epithelial cells both in vivo and in vitro. Methods Human corneal epithelial cells were isolated from the corneoscleral rims of male and female donors. Cells were processed either directly for RNA extraction, or first cultured in phenol red-free keratinocyte serum-free media. The RNA samples were examined for differentially expressed mRNAs by using of CodeLink Bioarrays and Affymetrix GeneChips. Data were analyzed with GeneSifter.Net software. Results Our results demonstrate that sex significantly influences the expression of over 600 genes in human corneal epithelial cells in vivo. These genes are involved in a broad spectrum of biologic processes, molecular functions and cellular components, such as metabolic processes, DNA replication, cell migration, RNA binding, oxidoreductase activity and nucleoli. We also identified significant, sex-related effects on gene expression in human corneal epithelial cells in vitro. However, with few exceptions (e.g., X- and Y-linked genes), these sex-related differences in gene expression in vitro were typically different than those in vivo. Conclusions Our findings support our hypothesis that sex-related differences exist in the gene expression of human corneal epithelial cells. Variations in gene expression may contribute to sex-related differences in the prevalence of certain corneal diseases. PMID:20011627

  7. 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.

  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. 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...

  10. 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...

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

    EPA Science Inventory

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

  12. 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...

  13. 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, ...

  14. Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn{sup 2+}

    SciTech Connect

    Tal, T.L.; Graves, L.M.; Silbajoris, R.; Bromberg, P.A.; Wu, W.; Samet, J.M. . E-mail: samet.james@epa.gov

    2006-07-01

    Epidemiological studies have implicated zinc (Zn{sup 2+}) 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 to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn{sup 2+}-induced EGFR activation in HAEC, we treated HAEC with 500 {mu}M ZnSO{sub 4} for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn{sup 2+} results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn{sup 2+}-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn{sup 2+} treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn{sup 2+}. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn{sup 2+} or V{sup 4+} was significantly diminished. Moreover, exposure of HAEC to Zn{sup 2+} also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn{sup 2+}-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn{sup 2+} exposure.

  15. LPS may enhance expression and release of HMGB1 in human nasal epithelial cells in vitro.

    PubMed

    Chen, D; Bellussi, L M; Passali, D; Chen, L

    2013-12-01

    Chronic rhinosinusitis with nasal polyps is a common disease with still unclear pathophysiologic mechanisms. The airway epithelial barrier has been shown to be involved in different chronic disorders, including rhinitis, nasal polyposis and asthma. High mobility group box 1 (HMGB1), a primarily nuclear protein, is involved in the induction of airway inflammation in patients with chronic rhinosinusitis, allergy, asthma and COPD. Pathogen-derived lipopolysaccharide is widely used as a trigger for inflammation. However, the molecular dialogue between LPS and HMGB1 in the delayed inflammatory processes remains to be explored, and the regulation of HMGB1 release through LPS from epithelial cells has not been extensively studied in patients with chronic rhinosinusitis and nasal polyps. The objective of the present study was to investigate the relocation of HMGB1 in LPS-induced human nasal epithelial cells in vitro. We obtained epithelial cells of nasal polyps from 10 patients requiring surgery for sinusitis at the ENT Department of the Chinese PLA General Hospital. The primary cultured human nasal epithelial (HNE) cells were stimulated with LPS. The expression and translocation of HMGB1 in intracellular and culture supernatants were determined using Western blot and immunofluorescence assay. HMGB1 protein was released in a time-dependent fashion in culture supernatants: in fact, expression of HMGB1 protein in HNE cells showed no significant changes at 0-24 h after exposure to 100 μg/ml LPS, but increased significantly at 48 and 72 hr. Immunofluorescence analysis revealed the transfer of HMGB1 from nuclei to cytoplasm in response to LPS exposure after 24 hr. These data reveal a hitherto unrecognized association between HMGB1 and LPS in human nasal epithelial cells. LPS can affect HMGB1 translocation and release, suggesting the involvement of HMGB1, through inflammatory mediators, in chronic rhinosinusitis with nasal polyps.

  16. 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

  17. 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

  18. Epithelial-mesenchymal transition and FOXA genes during tobacco smoke carcinogen induced transformation of human bronchial epithelial cells.

    PubMed

    Bersaas, Audun; Arnoldussen, Yke Jildouw; Sjøberg, Mari; Haugen, Aage; Mollerup, Steen

    2016-09-01

    Lung cancer is largely an environmentally caused disease with poor prognosis. An in vitro transformation model of human bronchial epithelial cells (HBEC) was used to study long-term effects of tobacco smoke carcinogens on epithelial-mesenchymal transition (EMT) and the forkhead box transcription factors FOXA1 and FOXA2. CDK4 and hTERT immortalized HBEC2 and HBEC12 cell lines were exposed weekly to either cigarette smoke condensate (CSC), benzo[a]pyrene, or methylnitrosourea. Transformed cell lines were established from soft-agar colonies after 12weeks of exposure. HBEC12 was transformed by all exposures while HBEC2 was only transformed by CSC. Untransformed HBEC2 showed little invasive capacity, whereas transformed cell lines completely closed the gap in a matrigel scratch wound assay. CDH1 was down-regulated in all of the transformed cell lines. In contrast, CDH2 was up-regulated in both HBEC2 and one of the HBEC12 transformed cell lines. Furthermore, transformed cells showed activation of EMT markers including SNAI1, ZEB1, VIM, and MMP2. All transformed cell lines had significant down-regulation of FOXA1 and FOXA2, indicating a possible role in cell transformation and EMT. ChIP analysis showed increased binding of Histone-H3 and macroH2A in FOXA1 and FOXA2 in the transformed HBEC2 cell lines, indicating a compact chromatin. In conclusion, long-term carcinogen exposure lead to down-regulation of FOXA1 and FOXA2 concomitantly with the occurrence of EMT and in vitro transformation in HBEC cells.

  19. Gli promotes epithelial-mesenchymal transition in human lung adenocarcinomas

    PubMed Central

    Jin, Joy Q.; Woodard, Gavitt A.; Tolani, Bhairavi; Luh, Thomas M.; Giroux-Leprieur, Etienne; Mo, Minli; Chen, Zhao; Che, Juanjuan; Zhang, Zhenfa; Zhou, Yong; Wang, Lei; Hao, Xishan; Jablons, David; Wang, Changli; He, Biao

    2016-01-01

    Adenocarcinoma is the most common type of lung cancer. Epithelial-mesenchymal transition (EMT) is required for tumor invasion/metastasis and the components that control this process are potential therapeutic targets. This study we examined the role of Gli in lung adenocarcinoma and whether its activation regulates metastasis through EMT in lung adenocarcinoma. We found that tumors with high Gli expression had significantly lower E-Cadherin expression in two independent cohorts of patients with lung adenocarcinoma that we studied. In vitro up-regulation of SHh resulted in increased cell migration while small molecule inhibitors of Smo or Gli significantly reduced cell mobility both in a wound healing assay and in a 3D cell invasion assay. Inhibition of Gli in vivo decreased tumor growth and induced an increase in E-Cadherin expression. Our results indicate that Gli may be critical for lung adenocarcinoma metastasis and that a novel Gli inhibitor shows promise as a therapeutic agent by preventing cell migration and invasion in vitro and significantly reducing tumor growth and increasing E-Cadherin expression in vivo. PMID:27533453

  20. A Case Report of Focal Epithelial Hyperplasia (Heck's disease) with PCR Detection of Human Papillomavirus.

    PubMed

    Ozden, Bora; Gunduz, Kaan; Gunhan, Omer; Ozden, Feyza Otan

    2011-12-01

    Focal epithelial hyperplasia or Heck's disease, is a rare viral infection of the oral mucosa caused by human papillomavirus. The frequency of this disease varies widely from one geographic region to another. In Caucasians there have been only few cases reported. This paper reports a case of focal epithelial hyperplasia and demonstrates the association with HPV subtype 32 through polymerase chain reaction (PCR) and sequencing of PCR products. A 7-year-old Caucasian girl was admitted to our clinic for investigation of multiple oral mucosal lesions in the mouth. Lesion was excised under local anesthesia without any complication. The lesion was diagnosed as focal epithelial hyperplasia according to both clinical and histopathological features. Dental staff should be aware of these kind of lesions and histopathological examination together with a careful clinical observation should be carried out for a definitive diagnosis.

  1. The Influence of 13-cis Retinoic Acid on Human Meibomian Gland Epithelial Cells

    PubMed Central

    Ding, Juan; Kam, Wendy R.; Dieckow, Julia; Sullivan, David A.

    2013-01-01

    Purpose. Meibomian gland dysfunction (MGD) is a primary cause of dry eye disease. One of the risk factors for MGD is exposure to 13-cis retinoic acid (13-cis RA), a metabolite of vitamin A. However, the mechanism is not well understood. We hypothesize that 13-cis RA inhibits cell proliferation, promotes cell death, alters gene and protein expressions, and attenuates cell survival pathways in human meibomian gland epithelial cells. Methods. To test our hypotheses, immortalized human meibomian gland epithelial cells were cultured with or without 13-cis RA for varying doses and time. Cell proliferation, cell death, gene expression, and proteins involved in proliferation/survival and inflammation were evaluated. Results. We found that 13-cis RA inhibited cell proliferation, induced cell death, and significantly altered the expression of 6726 genes, including those involved in cell proliferation, cell death, differentiation, keratinization, and inflammation, in human meibomian gland epithelial cells. Further, 13-cis RA also reduced the phosphorylation of Akt and increased the generation of interleukin-1β and matrix metallopeptidase 9. Conclusions. Exposure to 13-cis RA inhibits cell proliferation, increases cell death, alters gene expression, changes signaling pathways, and promotes inflammatory mediator and protease expression in meibomian gland epithelial cells. These effects may be responsible, at least in part, for the 13-cis RA–related induction of MGD. PMID:23722388

  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. Cigarette smoke extract reduces VEGF in primary human airway epithelial cells.

    PubMed

    Thaikoottathil, J V; Martin, R J; Zdunek, J; Weinberger, A; Rino, J G; Chu, H W

    2009-04-01

    Reduced vascular endothelial growth factor (VEGF) has been reported in bronchoalveolar lavage fluid and lungs of severe emphysema patients. Airway epithelial cells (AEC) are exposed to various environmental insults like cigarette smoke and bacterial infections, but their direct effect on VEGF production in well-differentiated primary human AEC remains unclear. The current authors determined the effect of cigarette smoke extract (CSE) alone and in combination with Mycoplasma pneumoniae (Mp) on VEGF production in well-differentiated primary normal human bronchial epithelial (NHBE) and small airway epithelial cells (SAEC) in air-liquid interface cultures. Secretion and expression of VEGF were determined by ELISA and real-time RT-PCR, respectively. Cell growth, apoptosis, extracellular signal-regulated kinase (ERK)1/2 and protein kinase (PK)C signalling pathways were evaluated to further dissect VEGF regulation under CSE treatment. CSE significantly reduced VEGF secretion in NHBE and SAEC. In SAEC, Mp alone significantly increased the VEGF, while the presence of CSE attenuated Mp-induced VEGF production. While ERK inhibitor reduced VEGF secretion only in NHBE, a PKC inhibitor significantly decreased VEGF secretion in both NHBE and SAEC. In conclusion, direct cigarette smoke extract exposure significantly reduced vascular endothelial growth factor production in well-differentiated primary human airway epithelial cells, in part through modifying extracellular signal-regulated kinase 1/2 and protein kinase C signalling pathways.

  4. Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells.

    PubMed

    Olsen, Colin E; Liguori, Andrew E; Zong, Yue; Lantz, R Clark; Burgess, Jefferey L; Boitano, Scott

    2008-08-01

    As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.

  5. Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function

    PubMed Central

    Bode, Lars; Salvestrini, Camilla; Park, Pyong Woo; Li, Jin-Ping; Esko, Jeffrey D.; Yamaguchi, Yu; Murch, Simon; Freeze, Hudson H.

    2007-01-01

    Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-γ, TNF-α, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate– or syndecan-1–deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-γ, TNF-α, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients. PMID:18064305

  6. Cadmium regulates the expression of the CFTR chloride channel in human airway epithelial cells.

    PubMed

    Rennolds, Jessica; Butler, Susie; Maloney, Kevin; Boyaka, Prosper N; Davis, Ian C; Knoell, Daren L; Parinandi, Narasimham L; Cormet-Boyaka, Estelle

    2010-07-01

    Cadmium is a toxic heavy metal ranked seventh on the Priority List of Hazardous Substances. As a byproduct of smelters, cadmium is a prevalent environmental contaminant. It is also a major component of cigarette smoke, and its inhalation is associated with decreased pulmonary function, lung cancer, and chronic obstructive pulmonary disease. Ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), play a central role in maintaining fluid homeostasis and lung functions. CFTR is mostly expressed in epithelial cells, and little is known about the effect of cadmium exposure on lung epithelial cell function. We show that exposure to cadmium decreases the expression of the CFTR protein and subsequent chloride transport in human airway epithelial cells in vitro. Impairment of CFTR protein expression was also observed in vivo in the lung of mice after intranasal instillation of cadmium. We established that the inhibitory effect of cadmium was not a nonspecific effect of heavy metals, as nickel had no effect on CFTR protein levels. Finally, we show that selected antioxidants, including alpha-tocopherol (vitamin E), but not N-acetylcysteine, can prevent the cadmium-induced suppression of CFTR. In summary, we have identified cadmium as a regulator of the CFTR chloride channel present in lung epithelial cells. Future strategies to prevent the deleterious effect of cadmium on epithelial cells and lung functions may benefit from the finding that alpha-tocopherol protects CFTR expression and function.

  7. Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

    PubMed Central

    Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

    Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery. PMID:25861018

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

    PubMed

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

    2012-12-01

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

  9. Transcription factor TCF4 maintains the properties of human corneal epithelial stem cells.

    PubMed

    Lu, Rong; Qu, Yangluowa; Ge, Jian; Zhang, Lili; Su, Zhitao; Pflugfelder, Stephen C; Li, De-Quan

    2012-04-01

    TCF4, a key transcription factor of Wnt signaling system, has been recently found to be essential for maintaining stem cells. However, its signaling pathway is not well elucidated. This study was to explore the functional roles and signaling pathway of TCF4 in maintaining adult stem cell properties using human corneal epithelial stem cells as a model. With immunofluorescent staining and real-time polymerase chain reaction, we observed that TCF4 was exclusively expressed in the basal layer of human limbal epithelium where corneal epithelial stem cells reside. TCF4 was found to be well colocalized with ABCG2 and p63, two recognized epithelial stem/progenitor cell markers. Using in vitro culture models of primary human corneal epithelial cells, we revealed that TCF4 mRNA and protein were upregulated by cells in exponential growth stage, and RNA interference by small interfering RNA-TCF4 (10-50 nM) transfection blocked TCF4 signaling and suppressed cell proliferation as measured by WST-1 assay. TCF4 silence was found to be accompanied by downregulated proliferation-associated factors p63 and survivin, as well as upregulated cyclin-dependent kinase inhibitor 1C (p57). By creating a wound healing model in vitro, we identified upregulation and activation of β-catenin/TCF4 with their protein translocation from cytoplasm to nuclei, as evaluated by reverse transcription-quantitative real-time polymerase chain reaction, immunostaining, and Western blotting. Upregulated p63/survivin and downregulated p57 were further identified to be TCF4 downstream molecules that promote cell migration and proliferation in wound healing process. These findings demonstrate that transcription factor TCF4 plays an important role in determining or maintaining the phenotype and functional properties of human corneal epithelial stem cells.

  10. Identification of common microRNA-mRNA regulatory biomodules in human epithelial cancers

    PubMed Central

    Yang, Xinan; Lee, Younghee; Fan, Hong; Sun, Xiao; Lussier, Yves A

    2010-01-01

    The complex regulatory network between microRNAs and gene expression remains unclear domain of active research. We proposed to address in part this complex regulation with a novel approach for the genome-wide identification of biomodules derived from paired microRNA and mRNA profiles, which could reveal correlations associated with a complex network of de-regulation in human cancer. Two published expression datasets for 68 samples with 11 distinct types of epithelial cancers and 21 samples of normal tissues were used, containing microRNA expression (Lu et al. Nature Letters 2005) and gene expression (Ramaswarmy et al. PNAS 2001) profiles, respectively. As results, the microRNA expression used jointly with mRNA expression can provide better classifiers of epithelial cancers against normal epithelial tissue than either dataset alone (p=1×10-10, F-Test). We identified a combination of six microRNA-mRNA biomodules that optimally classified epithelial cancers from normal epithelial tissue (total accuracy = 93.3%; 95% confidence intervals: 86% - 97%), using penalized logistic regression (PLR) algorithm and three-fold cross-validation. Three of these biomodules are individually sufficient to cluster epithelial cancers from normal tissue using mutual information distance. The biomodules contain 10 distinct microRNAs and 98 distinct genes, including well known tumor markers such as miR-15a, miR-30e, IRAK1, TGFBR2, DUSP16, CDC25B and PDCD2. In addition, there is a significant enrichment (Fisher’s exact test p=3×10-10) between putative microRNA-target gene pairs reported in five microRNA target databases and the inversely correlated micro-RNA-mRNA pairs in the biomodules. Further, microRNAs and genes in the biomodules were found in abstracts mentioning epithelial cancers (Fisher Exact Test, unadjusted p<0.05). Taken together, these results strongly suggest that the discovered microRNA-mRNA biomodules correspond to regulatory mechanisms common to human epithelial cancer

  11. F-actin binding protein, anillin, regulates integrity of intercellular junctions in human epithelial cells

    PubMed Central

    Feygin, Alex; Ivanov, Andrei I.

    2015-01-01

    Tight junctions (TJ) and adherens junctions (AJ) are key morphological features of differentiated epithelial cells that regulate the integrity and permeability of tissue barriers. Structure and remodeling of epithelial junctions depends on their association with the underlying actomyosin cytoskeleton. Anillin is a unique scaffolding protein interacting with different cytoskeletal components, including actin filaments and myosin motors. Its role in the regulation of mammalian epithelial junctions remains unexplored. Downregulation of anillin expression in human prostate, colonic, and lung epithelial cells triggered AJ and TJ disassembly without altering the expression of junctional proteins. This junctional disassembly was accompanied by dramatic disorganization of the perijunctional actomyosin belt; while the general architecture of the actin cytoskeleton, and activation status of non-muscle myosin II, remained unchanged. Furthermore, loss of anillin disrupted the adducin-spectrin membrane skeleton at the areas of cell-cell contact, selectively decreased γ-adducin expression, and induced cytoplasmic aggregation of αII-spectrin. Anillin knockdown activated c-Jun N-terminal kinase (JNK), and JNK inhibition restored AJ and TJ integrity and cytoskeletal organization in anillin-depleted cells. These findings suggest a novel role for anillin in regulating intercellular adhesion in model human epithelia by mechanisms involving the suppression of JNK activity and controlling the assembly of the perijunctional cytoskeleton. PMID:25809162

  12. Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

    PubMed Central

    Gruenert, D C; Basbaum, C B; Welsh, M J; Li, M; Finkbeiner, W E; Nadel, J A

    1988-01-01

    To facilitate understanding of the mechanisms underlying pulmonary diseases, including lung cancer and cystic fibrosis, we have transformed and characterized cultures of human tracheal epithelial cells. Cells were transfected by calcium phosphate precipitation with a plasmid containing a replication-defective simian virus 40 (SV40) genome. Colonies of cells with enhanced growth potential were isolated and analyzed for transformation- and epithelial-specific characteristics. Precrisis cells were observed to express the SV40 large tumor antigen, produce cytokeratins, have microvilli, and form tight junctions. After crisis, cells continued to express the SV40 large tumor antigen as well as epithelial-specific cytokeratins and to display the apical membrane microvilli. Apical membrane Cl channels were opened in postcrisis cells exposed to 50 microM forskolin. These channels showed electrical properties similar to those observed in primary cultures. The postcrisis cells have been in culture for greater than 250 generations and are potentially "immortal." In addition to providing a useful in vitro model for the study of ion transport by human airway epithelial cells, the cells can be used to examine stages of neoplastic progression. Images PMID:2457904

  13. Role of pili in adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells.

    PubMed Central

    Doig, P; Todd, T; Sastry, P A; Lee, K K; Hodges, R S; Paranchych, W; Irvin, R T

    1988-01-01

    The ability of pili from Pseudomonas aeruginosa K (PAK) to act as an adhesin to human respiratory epithelial cells was examined using an in vitro adhesion assay. Equilibrium analysis of PAK binding to human buccal epithelial cells (BECs) and tracheal epithelial cells (TECs) by means of a Langmuir adsorption isotherm revealed that the maximum numbers of binding sites per epithelial cell (N) were 255 for BECs and 236 for TECs, with apparent association constants (Ka) of 2.8 x 10(-9) and 5.8 x 10(-9) ml/CFU, respectively. Trypsinization of the BECs before the binding assay increased N to 605 and decreased the Ka to 1.7 x 10(-9) ml/CFU. Addition of homologous pili to the binding assay with BECs or TECs or the addition of anti-pilus Fab fragments inhibited PAK adherence. Binding of purified pili to BECs was shown to reach saturation. Purified pili and PAK competed for the same receptor on the BEC surface. Further, by using peptide fragments of PAK pilin (derived from the native pili or produced synthetically) in the binding assay for PAK to BECs, we have presumptively identified the pilus binding domain in the C-terminal region of the pilin and shown that the C-terminal disulfide bridge is important in maintaining the functionality of the binding domain. PMID:2897336

  14. TGF-beta during human colorectal carcinogenesis: the shift from epithelial to mesenchymal signaling.

    PubMed

    Matsuzaki, K; Seki, T; Okazaki, K

    2006-12-01

    Transforming growth factor-beta (TGF-beta) activates not only TGF-beta type I receptor (Tbeta RI) but also c-Jun N-terminal kinase (JNK), converting the mediator Smad3 to two distinct phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). While Tbeta RI/pSmad3C pathway inhibits growth of normal epithelial cells, the activated mesenchymal cells invade via JNK/pSmad3L pathway. During sporadic human colorectal carcinogenesis, TGF-beta signaling confers a selective advantage upon tumor cells by shifting from epithelial Tbeta RI/pSmad3C pathway to mesenchymal JNK/pSmad3L pathway. Loss of epithelial homeostasis and acquisition of a migratory, mesenchymal phenotype are essential for tumor invasion. In a future, specific inhibition of the JNK/pSmad3L pathway will become a therapy for human colorectal cancer that restores the lost tumor-suppressive function observed in normal colorectal epithelial cells at the expense of effects promoting the aggressive behavior.

  15. Hormone Production by Epithelial Cells of Human Thymus in vitro.

    PubMed

    Yarilin, A. A.; Sharova, N. I.; Bulanova, E. C.; Kotchergina, N. I.; Mitin, A. N.; Kharchenko, T. Yu.; Arshinov, V. Yu.

    1996-12-01

    The conditions of hormone production by human thymic stromal cell line were studied. Human thymic stromal cells did not produce any hormones in 5-day monoculture. Co-cultivation of these cells with human thymocytes induced alpha1-thymosin and thymulin production increased to 4-5 days of co-cultivation. An increase in number of human thymic stromal cells and thymocyte elimination were observed in co-culture. The maximal stimulation of proliferation and hormone secretion by human thymic stromal cell was reached in their co-culturing with thymocytes at relative concentrations of 10(4) and 10(7) cells per ml. Thymocyte viability was important for inducing the stimulatory effect. The effect of viable cells could not be replaced by their supernatant. Stimulatory activity of CD4(-)CD8(-) and CD4(+)CD8(+) thymocytes was comparable, alpha1-thymosin and some of its synthetic fragments did not influence alpha1-thymosin synthesis or slightly inhibited it (in high concentrations). Synthetic peptide corresponding to C-terminal half of alpha1-thymosin molecule strongly enhanced production of this hormone.

  16. A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells.

    PubMed Central

    Cunningham, A C; Zhang, J G; Moy, J V; Ali, S; Kirby, J A

    1997-01-01

    Human lung alveolar epithelial cells constitutively express class II major histocompatibility complex (MHC). Human lung microvascular endothelial and small airway epithelial cells can be induced to express class II MHC by stimulation with the pro-inflammatory cytokine interferon-gamma. The levels of class II MHC on lung epithelial and endothelial cells were comparable to those seen on an Epstein-Barr virus (EBV)-transformed B-cell line. However, the costimulatory molecules B7-1 and B7-2 were not expressed. The ability of the class II MHC expressing human lung parenchymal cells to present alloantigen to CD4+ T lymphocytes was investigated. Freshly isolated human alveolar epithelial cells (type II pneumocytes) and monolayers of interferon-gamma-stimulated small airway epithelial and lung microvascular endothelial cells were co-cultured with allogeneic CD4+ T lymphocytes and proliferation determined by [3H]thymidine incorporation. A clear difference was observed between effects of the epithelial and endothelial cells on CD4+ T-lymphocyte activation. Alveolar and small airway epithelial cells failed to stimulate the proliferation of allogeneic CD4+ T lymphocytes whereas lung microvascular endothelial cells did stimulate proliferation. This difference could not be explained by the levels of class II MHC or the lack of B7-1 and B7-2 solely. Microvascular endothelial cells, and not alveolar or small airway epithelial cells, possess B7-independent costimulatory pathways. PMID:9301537

  17. Biodegradable Gelatin Microcarriers Facilitate Re-Epithelialization of Human Cutaneous Wounds - An In Vitro Study in Human Skin

    PubMed Central

    Lönnqvist, Susanna; Rakar, Jonathan; Briheim, Kristina; Kratz, Gunnar

    2015-01-01

    The possibility to use a suspended tridimensional matrix as scaffolding for re-epithelialization of in vitro cutaneous wounds was investigated with the aid of a human in vitro wound healing model based on viable full thickness skin. Macroporous gelatin microcarriers, CultiSpher-S, were applied to in vitro wounds and cultured for 21 days. Tissue sections showed incorporation of wound edge keratinocytes into the microcarriers and thicker neoepidermis in wounds treated with microcarriers. Thickness of the neoepidermis was measured digitally, using immunohistochemical staining of keratins as epithelial demarcation. Air-lifting of wounds enhanced stratification in control wounds as well as wounds with CultiSpher-S. Immunohistochemical staining revealed expression of keratin 5, keratin 10, and laminin 5 in the neoepidermal component. We conclude that the CultiSpher-S microcarriers can function as tissue guiding scaffold for re-epithelialization of cutaneous wounds. PMID:26061630

  18. Biodegradable Gelatin Microcarriers Facilitate Re-Epithelialization of Human Cutaneous Wounds - An In Vitro Study in Human Skin.

    PubMed

    Lönnqvist, Susanna; Rakar, Jonathan; Briheim, Kristina; Kratz, Gunnar

    2015-01-01

    The possibility to use a suspended tridimensional matrix as scaffolding for re-epithelialization of in vitro cutaneous wounds was investigated with the aid of a human in vitro wound healing model based on viable full thickness skin. Macroporous gelatin microcarriers, CultiSpher-S, were applied to in vitro wounds and cultured for 21 days. Tissue sections showed incorporation of wound edge keratinocytes into the microcarriers and thicker neoepidermis in wounds treated with microcarriers. Thickness of the neoepidermis was measured digitally, using immunohistochemical staining of keratins as epithelial demarcation. Air-lifting of wounds enhanced stratification in control wounds as well as wounds with CultiSpher-S. Immunohistochemical staining revealed expression of keratin 5, keratin 10, and laminin 5 in the neoepidermal component. We conclude that the CultiSpher-S microcarriers can function as tissue guiding scaffold for re-epithelialization of cutaneous wounds.

  19. Epithelial glycoprotein-2 expression is subject to regulatory processes in epithelial-mesenchymal transitions during metastases: an investigation of human cancers transplanted into severe combined immunodeficient mice.

    PubMed

    Jojović, M; Adam, E; Zangemeister-Wittke, U; Schumacher, U

    1998-10-01

    The human cell-surface antigen epithelial glycoprotein-2 recognized by the monoclonal antibody MOC-31 is an epithelial tumour-associated glycoprotein expressed in non-squamous carcinomas. MOC-31 immunoreactivity was investigated in human breast, colon, ovarian and lung cancer cell lines, grown either in vitro or in severe combined immunodeficient (SCID) mice as solid tumours and/or metastases. Three of four small-cell lung cancer cell lines (NCI-H69, OH3 and SW2) and three of four ovarian cancer cell lines (SoTu 1, 3 and 4) expressed epithelial glycoprotein-2. In contrast, all three breast (MCF-7, BT20, T47D) and all three colon (HT29, CACO2, SW480) cancer cell lines strongly reacted with monoclonal antibody MOC-31. A notable difference in MOC-31 immunoreactivity was observed in spontaneously formed lung metastases of HT29 colon cancer cells. Whereas larger metastases (> 30 cells) reacted with a similar staining pattern to the primary tumour, smaller metastases did not. These findings indicate that differentiation processes during the epithelial-mesenchymal transition occur in metastases, which lead to a transient loss of epithelial glycoprotein-2 expression during the migratory and early post-migratory period. This loss of antigen expression indicates that the process of metastases formation is a regulatory event, and this transient loss of antigen expression might represent a potential obstacle to antibody-based therapy in the setting of minimal residual disease.

  20. Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells

    PubMed Central

    Belloy, Marcy; Saulnier-Blache, Jean-Sébastien; Casemayou, Audrey; Ducasse, Laure; Grès, Sandra; Bellière, Julie; Caubet, Cécile; Bascands, Jean-Loup; Schanstra, Joost P.; Buffin-Meyer, Bénédicte

    2015-01-01

    Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, β-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium. PMID:26146837

  1. Inhibitory effects of carbocisteine on type A seasonal influenza virus infection in human airway epithelial cells.

    PubMed

    Yamaya, Mutsuo; Nishimura, Hidekazu; Shinya, Kyoko; Hatachi, Yukimasa; Sasaki, Takahiko; Yasuda, Hiroyasu; Yoshida, Motoki; Asada, Masanori; Fujino, Naoya; Suzuki, Takaya; Deng, Xue; Kubo, Hiroshi; Nagatomi, Ryoichi

    2010-08-01

    Type A human seasonal influenza (FluA) virus infection causes exacerbations of bronchial asthma and chronic obstructive pulmonary disease (COPD). l-carbocisteine, a mucolytic agent, reduces the frequency of common colds and exacerbations in COPD. However, the inhibitory effects of l-carbocisteine on FluA virus infection are uncertain. We studied the effects of l-carbocisteine on FluA virus infection in airway epithelial cells. Human tracheal epithelial cells were pretreated with l-carbocisteine and infected with FluA virus (H(3)N(2)). Viral titers in supernatant fluids, RNA of FluA virus in the cells, and concentrations of proinflammatory cytokines in supernatant fluids, including IL-6, increased with time after infection. l-carbocisteine reduced viral titers in supernatant fluids, RNA of FluA virus in the cells, the susceptibility to FluA virus infection, and concentrations of cytokines induced by virus infection. The epithelial cells expressed sialic acid with an alpha2,6-linkage (SAalpha2,6Gal), a receptor for human influenza virus on the cells, and l-carbocisteine reduced the expression of SAalpha2,6Gal. l-carbocisteine reduced the number of acidic endosomes from which FluA viral RNA enters into the cytoplasm and reduced the fluorescence intensity from acidic endosomes. Furthermore, l-carbocisteine reduced NF-kappaB proteins including p50 and p65 in the nuclear extracts of the cells. These findings suggest that l-carbocisteine may inhibit FluA virus infection, partly through the reduced expression of the receptor for human influenza virus in the human airway epithelial cells via the inhibition of NF-kappaB and through increasing pH in endosomes. l-carbocisteine may reduce airway inflammation in influenza virus infection.

  2. Staphylococcus aureus Infection Reduces Nutrition Uptake and Nucleotide Biosynthesis in a Human Airway Epithelial Cell Line

    PubMed Central

    Gierok, Philipp; Harms, Manuela; Methling, Karen; Hochgräfe, Falko; Lalk, Michael

    2016-01-01

    The Gram positive opportunistic human pathogen Staphylococcus aureus induces a variety of diseases including pneumonia. S. aureus is the second most isolated pathogen in cystic fibrosis patients and accounts for a large proportion of nosocomial pneumonia. Inside the lung, the human airway epithelium is the first line in defence with regard to microbial recognition and clearance as well as regulation of the immune response. The metabolic host response is, however, yet unknown. To address the question of whether the infection alters the metabolome and metabolic activity of airway epithelial cells, we used a metabolomics approach. The nutrition uptake by the human airway epithelial cell line A549 was monitored over time by proton magnetic resonance spectroscopy (1H-NMR) and the intracellular metabolic fingerprints were investigated by gas chromatography and high performance liquid chromatography (GC-MS) and (HPLC-MS). To test the metabolic activity of the host cells, glutamine analogues and labelled precursors were applied after the infection. We found that A549 cells restrict uptake of essential nutrients from the medium after S. aureus infection. Moreover, the infection led to a shutdown of the purine and pyrimidine synthesis in the A549 host cell, whereas other metabolic routes such as the hexosamine biosynthesis pathway remained active. In summary, our data show that the infection with S. aureus negatively affects growth, alters the metabolic composition and specifically impacts the de novo nucleotide biosynthesis in this human airway epithelial cell model. PMID:27834866

  3. Matrigel-induced tubular morphogenesis of human eccrine sweat gland epithelial cells.

    PubMed

    Lei, Xia; Liu, Bo; Wu, Jinjin; Lu, Yuangang; Yang, Yadong

    2011-09-01

    Human eccrine sweat glands are tubule-structured glands of the skin that are vital in thermoregulation, secretion, and excretion of water and electrolytes. A study of tubular morphogenesis in vitro would facilitate the development of a tissue engineering model for eccrine sweat glands and other tubule-structured glands. Matrigel, a basement membrane matrix, has been shown to promote differentiation and morphogenesis of many different cell types, including tubular cells. This study investigated the growth, differentiation, and tubular morphogenesis of human eccrine sweat gland epithelial cells cultured in Matrigel. Human eccrine gland epithelial cells were isolated and cultured in vitro. The cell growth in Matrigel was evidenced by the formation of cell clusters, which were observed under an inverted microscope. The internal structure of the cell clusters was further investigated by hematoxylin-eosin (HE) staining and confocal laser scanning microscopy (CLSM) of propidium iodide-stained nuclei. The results demonstrated that although on a plastic surface or in a collagen gel the cells could not form tubular structures, they formed tubular structures when cultured in Matrigel. Consequently, we conclude that Matrigel can promote tubular morphogenesis of human eccrine sweat gland epithelial cells.

  4. Functional expression of nicotine influx transporter in A549 human alveolar epithelial cells.

    PubMed

    Tega, Yuma; Yuzurihara, Chihiro; Kubo, Yoshiyuki; Akanuma, Shin-ichi; Ehrhardt, Carsten; Hosoya, Ken-ichi

    2016-02-01

    Nicotine is a potent addictive alkaloid, and is rapidly absorbed through the alveoli of the lung. However, the transport mechanism of nicotine at the human alveolar epithelial barrier has not been investigated in great detail. In the present study, the transport mechanism of nicotine across alveolar epithelium was investigated in vitro using A549 cells, a human adenocarcinoma-derived cell line with an alveolar epithelial cell like phenotype. Nicotine uptake by A549 cells exhibited time-, temperature-, and concentration-dependence with a Km of 50.4 μM. These results suggest that a carrier-mediated transport process is involved in nicotine transport in human alveolar epithelial cells. Nicotine uptake by A549 cells was insensitive to change in extracellular pH. Moreover, nicotine uptake by A549 cells could be inhibited by organic cations such as verapamil and pyrilamine, but not typical substrates of organic cation transporters and β2-agonist. These results suggest that a novel, not yet molecularly identified, organic cation transporter plays a role in nicotine transport which is unlikely to interact with β2-agonist transport. This nicotine influx transporter in human alveolar epithelium might have implications for the rapid absorption of nicotine into the systemic circulation.

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

    SciTech Connect

    Weng, Yu-I; Hsu, Pei-Yin; Liyanarachchi, Sandya; Liu, Joseph; Deatherage, Daniel E.; Huang Yiwen; Zuo Tao; Rodriguez, Benjamin; Lin, Ching-Hung; Cheng, Ann-Lii; Huang, Tim H.-M.

    2010-10-15

    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 expression 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.

  6. Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries.

    PubMed

    Rochon, Marie-Hélène; Fradette, Julie; Fortin, Véronique; Tomasetig, Florence; Roberge, Charles J; Baker, Kathleen; Berthod, François; Auger, François A; Germain, Lucie

    2010-05-01

    The survival of thick tissues/organs produced by tissue engineering requires rapid revascularization after grafting. Although capillary-like structures have been reconstituted in some engineered tissues, little is known about the interaction between normal epithelial cells and endothelial cells involved in the in vitro angiogenic process. In the present study, we used the self-assembly approach of tissue engineering to examine this relationship. An endothelialized tissue-engineered dermal substitute was produced by adding endothelial cells to the tissue-engineered dermal substitute produced by the self-assembly approach. The latter consists in culturing fibroblasts in the medium supplemented with serum and ascorbic acid. A network of tissue-engineered capillaries (TECs) formed within the human extracellular matrix produced by dermal fibroblasts. To determine whether epithelial cells modify TECs, the size and form of TECs were studied in the endothelialized tissue-engineered dermal substitute cultured in the presence or absence of epithelial cells. In the presence of normal keratinocytes from skin, cornea or uterine cervix, endothelial cells formed small TECs (cross-sectional area estimated at less than 50 microm(2)) reminiscent of capillaries found in the skin's microcirculation. In contrast, TECs grown in the absence of epithelial cells presented variable sizes (larger than 50 microm(2)), but the addition of keratinocyte-conditioned media or exogenous vascular endothelial growth factor induced their normalization toward a smaller size. Vascular endothelial growth factor neutralization inhibited the effect of keratinocyte-conditioned media. These results provide new direct evidence that normal human epithelial cells play a role in the regulation of the underlying TEC network, and advance our knowledge in tissue engineering for the production of TEC networks in vitro.

  7. 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.

  8. Promoting effect of lactoferrin on barrier function and epithelial differentiation of human keratinocytes.

    PubMed

    Uchida, Ryo; Aoki, Reiji; Aoki-Yoshida, Ayako; Tajima, Atsushi; Takayama, Yoshiharu

    2017-02-01

    The purpose of this study was to elucidate the effects of bovine lactoferrin on keratinocyte differentiation and barrier function. Addition of bovine lactoferrin to differentiating HaCaT human keratinocytes led to increased transepithelial electrical resistance (TER), a marker of epithelial barrier function. This elevation was followed by upregulation of two differentiation markers, involucrin and filaggrin. The expression level of sterol regulatory element-binding protein-1 was also enhanced by bovine lactoferrin. The lactoferrin-induced upregulation of involucrin and filaggrin expression were confirmed in normal human epidermal keratinocytes (NHEK). Treatment with SB203580, a p38 mitogen-activated protein kinase (MAPK) α inhibitor, impaired the upregulation of involucrin and filaggrin expression in response to lactoferrin. The elevation of p38 MAPK phosphorylation was further enhanced by lactoferrin in the initial stage of differentiation of HaCaT keratinocytes. The findings suggest that bovine lactoferrin promotes epithelial differentiation by a p38-MAPK-dependent mechanism.

  9. Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells.

    PubMed

    Kolesnikova, Larissa; Heck, Sonja; Matrosovich, Tatyana; Klenk, Hans-Dieter; Becker, Stephan; Matrosovich, Mikhail

    2013-05-01

    The epithelium of conducting airways represents the main target for influenza virus in mammals. However, the peculiarities of virus interactions with differentiated airway epithelial cells remain largely unknown. Here, influenza virus budding was studied in differentiated cultures of human tracheobronchial epithelial cells using transmission electron microscopy. Budding of spherical and filamentous virions was observed on the apical surfaces of cells with no association with cilia and secretory granules. Quantitative analysis of the distribution of viral buds on the cell surface indicated that the tips of the microvilli represented a prominent site of influenza virus budding in the human airway epithelium. As the microvilli of differentiated cells are involved in many fundamental cell functions, these data will prompt further studies on the biological significance of microvilli-associated budding for virus replication, transmission and pathogenicity.

  10. Epithelial-connective tissue boundary in the oral part of the human soft palate

    PubMed Central

    PAULSEN, FRIEDRICH; THALE, ANDREAS

    1998-01-01

    The papillary layer of the oral part of the human soft palate was studied in 31 subjects of different age by means of histological, immunohistochemical and scanning electron microscopical methods. For scanning electron microscopy a new maceration method was introduced. Results determine epithelial thickness, height and density of connective tissue papillae and their 3-dimensional architecture inside the lining epithelium as well as the collagenous arrangement of the openings of the glandular ducts. The individual connective tissue papillae of the soft palate are compared with the connective tissue boundary on the other side of the oral cavity. The connective tissue plateaux carrying a variable number of connective tissue papillae were found to be the basic structural units of the papillary body. The function of the epithelial-connective tissue interface and the extracellular matrix arrangement in the lamina propria are discussed in order to promote the comparability of normal with pathologically altered human soft palates. PMID:9877301

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

    PubMed Central

    Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A; Costa, Max

    2016-01-01

    The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study , we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA – mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. PMID:26780400

  12. 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.

  13. Human turbinate mesenchymal stromal cell sheets with bellows graft for rapid tracheal epithelial regeneration.

    PubMed

    Park, Jeong Hun; Park, Ju Young; Nam, Inn-Chul; Hwang, Se-Hwan; Kim, Choung-Soo; Jung, Jin Woo; Jang, Jinah; Lee, Hyungseok; Choi, Yeongjin; Park, Sun Hwa; Kim, Sung Won; Cho, Dong-Woo

    2015-10-01

    Rapid functional epithelial regeneration on the luminal surface is essential when using artificial tracheal grafts to repair tracheal defects. In this study, we imposed human turbinate mesenchymal stromal cell (hTMSC) sheets for tracheal epithelial regeneration, and then assessed their potential as a new clinical cell source. In vitro, hTMSCs sheets showed high capacity to differentiate into tracheal epithelium. We fabricated a poly(ε-caprolactone) (PCL) tracheal graft by indirect three-dimensional (3D) printing technique and created a composite construct by transplanting the hTMSC sheets to its luminal surface of the tracheal graft, then applied this tissue-engineered tracheal graft to non-circumferential tracheal reconstruction in a rabbit model. 4 weeks after implantation, the luminal surface of tissue-engineered tracheal graft was covered by a mature and highly-ciliated epithelium, whereas tracheal grafts without hTMSC sheets were covered by only a thin, immature epithelium. Therefore, hTMSC sheets on the luminal surface of a tissue-engineered tracheal graft can accelerate the tracheal epithelial regeneration, and the tissue-engineered tracheal graft with hTMSC sheets provides a useful clinical alternative for tracheal epithelial regeneration.

  14. Neural differentiation of choroid plexus epithelial cells: role of human traumatic cerebrospinal fluid

    PubMed Central

    Hashemi, Elham; Sadeghi, Yousef; Aliaghaei, Abbas; Seddighi, Afsoun; Piryaei, Abbas; Broujeni, Mehdi Eskandarian; Shaerzadeh, Fatemeh; Amini, Abdollah; Pouriran, Ramin

    2017-01-01

    As the key producer of cerebrospinal fluid (CSF), the choroid plexus (CP) provides a unique protective system in the central nervous system. CSF components are not invariable and they can change based on the pathological conditions of the central nervous system. The purpose of the present study was to assess the effects of non-traumatic and traumatic CSF on the differentiation of multipotent stem-like cells of CP into the neural and/or glial cells. CP epithelial cells were isolated from adult male rats and treated with human non-traumatic and traumatic CSF. Alterations in mRNA expression of Nestin and microtubule-associated protein (MAP2), as the specific markers of neurogenesis, and astrocyte marker glial fibrillary acidic protein (GFAP) in cultured CP epithelial cells were evaluated using quantitative real-time PCR. The data revealed that treatment with CSF (non-traumatic and traumatic) led to increase in mRNA expression levels of MAP2 and GFAP. Moreover, the expression of Nestin decreased in CP epithelial cells treated with non-traumatic CSF, while treatment with traumatic CSF significantly increased its mRNA level compared to the cells cultured only in DMEM/F12 as control. It seems that CP epithelial cells contain multipotent stem-like cells which are inducible under pathological conditions including exposure to traumatic CSF because of its compositions. PMID:28250752

  15. Clinicopathology of EpCAM and EGFR in Human Epithelial Ovarian Carcinoma

    PubMed Central

    Zheng, Jingying; Zhao, Lijing; Wang, Yi; Zhao, Shuhua; Cui, Manhua

    2017-01-01

    Abstract The objective of this study was to explore the expression of EpCAM and EGFR in human epithelial ovarian cancer (EOC) and their correlation with clinicopathological parameters. The protein expression levels of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) were evaluated by immunohistochemistry in formalin-fixed paraffin-embedded specimens from 30 patients with epithelial ovarian carcinoma and 15 normal ovary tissues. Clinicopathological characteristics were gathered by retrospective review of the patients’ files. The correlation between EpCAM and EGFR expression, as well as their association with clinical pathological parameters were investigated. The SPSS 17.0 package was used to perform statistical analyses. The positive expression rates of EpCAM and EGFR were significantly elevated in epithelial ovarian cancer tissues than in normal ovary tissues. The positive expressions of EpCAM and EGFR in EOC were associated with International Federation of Gynecology and Obstetrics (FIGO) stage and tumor differentiation, lymph node metastasis. Spearman correlation analysis demonstrated a significant positive association between EpCAM and EGFR expression in EOC. The co-expression of EpCAM and EGFR may play an important role in the carcinogenesis of EOC and might provide a promising molecular therapeutic target.

  16. Stress signaling from human mammary epithelial cells contributes to phenotypes of mammographic density.

    PubMed

    DeFilippis, Rosa Anna; Fordyce, Colleen; Patten, Kelley; Chang, Hang; Zhao, Jianxin; Fontenay, Gerald V; Kerlikowske, Karla; Parvin, Bahram; Tlsty, Thea D

    2014-09-15

    Telomere malfunction and other types of DNA damage induce an activin A-dependent stress response in mortal nontumorigenic human mammary epithelial cells that subsequently induces desmoplastic-like phenotypes in neighboring fibroblasts. Some characteristics of this fibroblast/stromal response, such as reduced adipocytes and increased extracellular matrix content, are observed not only in tumor tissues but also in disease-free breast tissues at high risk for developing cancer, especially high mammographic density tissues. We found that these phenotypes are induced by repression of the fatty acid translocase CD36, which is seen in desmoplastic and disease-free high mammographic density tissues. In this study, we show that epithelial cells from high mammographic density tissues have more DNA damage signaling, shorter telomeres, increased activin A secretion and an altered DNA damage response compared with epithelial cells from low mammographic density tissues. Strikingly, both telomere malfunction and activin A expression in epithelial cells can repress CD36 expression in adjacent fibroblasts. These results provide new insights into how high mammographic density arises and why it is associated with breast cancer risk, with implications for the definition of novel invention targets (e.g., activin A and CD36) to prevent breast cancer.

  17. TIMP-1 via TWIST1 Induces EMT Phenotypes in Human Breast Epithelial Cells

    PubMed Central

    D’Angelo, Rosemarie Chirco; Liu, Xu-Wen; Najy, Abdo J.; Jung, Young Suk; Won, Joshua; Chai, Karl X.; Fridman, Rafael; Kim, Hyeong-Reh Choi

    2014-01-01

    Tissue inhibitor of metalloproteinase-1 (TIMP1) regulates intracellular signaling networks for inhibition of apoptosis. Tetraspanin (CD63), a cell surface binding partner for TIMP-1, was previously shown to regulate integrin-mediated survival pathways in the human breast epithelial cell line MCF10A. In the current study, we show that TIMP-1 expression induces phenotypic changes in cell morphology, cell adhesion, cytoskeletal remodeling, and motility, indicative of an epithelial-mesenchymal transition (EMT). This is evidenced by loss of the epithelial cell adhesion molecule E-cadherin with an increase in the mesenchymal markers vimentin, N-cadherin, and fibronectin. Signaling through TIMP-1, but not TIMP-2, induces the expression of TWIST1, an important EMT transcription factor known to suppress E-cadherin transcription, in a CD63-dependent manner. RNAi-mediated knockdown of TWIST1 rescued E-cadherin expression in TIMP-1 overexpressing cells, demonstrating a functional significance of TWIST1 in TIMP-1 mediated EMT. Furthermore, analysis of TIMP-1 structural mutants reveals that TIMP-1 interactions with CD63 that activate cell survival signaling and EMT do not require the MMP-inhibitory domain of TIMP-1. Taken together, these data demonstrate that TIMP-1 binding to CD63 activates intracellular signal transduction pathways, resulting in EMT-like changes in breast epithelial cells, independent of its MMP-inhibitory function. PMID:24895412

  18. Expression of IL-4/IL-13 receptors in differentiating human airway epithelial cells

    PubMed Central

    Martin, Linda D.; Stern, Randi; Laxman, Bharathi; Marroquin, Bertha A.

    2010-01-01

    IL-4 and IL-13 elicit several important responses in airway epithelium including chemokine secretion and mucous secretion that may contribute to airway inflammation, cell migration, and differentiation. These cytokines have overlapping but not identical effector profiles likely due to shared subunits in their receptor complexes. These receptors are variably described in epithelial cells, and the relative expression, localization, and function of these receptors in differentiated and repairing epithelial cells are not clear. We examined IL-4/IL-13 receptor expression and localization in primary airway epithelial cells collected from normal human lungs and grown under conditions yielding both undifferentiated and differentiated cells inclusive of basal, goblet, and ciliated cell phenotypes. Gene expression of the IL-4Rα, IL-2Rγc, IL-13Rα1, and IL-13Rα2 receptor subunits increased with differentiation, but different patterns of localization and protein abundance were seen for each subunit based on both differentiation and the cell subtypes present. Increased expression of receptor subunits observed in more differentiated cells was associated with more substantial functional responses to IL-4 stimulation including increased eotaxin-3 expression and accelerated migration after injury. We demonstrate substantial differences in IL-4/IL-13 receptor subunit expression and responsiveness to IL-4 based on the extent of airway epithelial cell differentiation and suggest that these differences may have functional consequences in airway inflammation. PMID:20729386

  19. Stress Signaling from Human Mammary Epithelial Cells Contributes to Phenotypes of Mammographic Density

    PubMed Central

    Patten, Kelley; Chang, Hang; Zhao, Jianxin; Fontenay, Gerald V.; Kerlikowske, Karla; Parvin, Bahram; Tlsty, Thea D.

    2014-01-01

    Telomere malfunction and other types of DNA damage induce an activin A-dependent stress response in mortal non-tumorigenic human mammary epithelial cells that subsequently induces desmoplastic-like phenotypes in neighboring fibroblasts. Some characteristics of this fibroblast/stromal response, such as reduced adipocytes and increased extracellular matrix content, are observed not only in tumor tissues but also in disease-free breast tissues at high risk for developing cancer, especially high mammographic density tissues. We found that these phenotypes are induced by repression of the fatty acid translocase CD36, which is seen in desmoplastic and disease-free high mammographic density tissues. In this study, we show that epithelial cells from high mammographic density tissues have more DNA damage signaling, shorter telomeres, increased activin A secretion and an altered DNA damage response compared to epithelial cells from low mammographic density tissues. Strikingly, both telomere malfunction and activin A expression in epithelial cells can repress CD36 expression in adjacent fibroblasts. These results provide new insights into how high mammographic density arises and why it is associated with breast cancer risk, with implications for the definition of novel invention targets (e.g. activin A, CD36) to prevent breast cancer. PMID:25172842

  20. Focal epithelial hyperplasia (Heck's disease): report of two cases with PCR detection of human papillomavirus DNA.

    PubMed

    Jayasooriya, P R; Abeyratne, S; Ranasinghe, A W; Tilakaratne, W M

    2004-07-01

    Focal epithelial hyperplasia (FEH) (Heck's disease) is essentially a benign oral infection produced by the human papillomavirus (HPV). Although this condition is known to exist in numerous populations and ethnic groups, it is relatively rare in South-East Asia. The following report is based on two cases of adult FEH with histopathological features in favour of the disease. In addition, polymerase chain reaction was performed to detect the presence of HPV DNA in the lesions in order to confirm the histopathological diagnosis.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed Central

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

    1982-01-01

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

  3. Tsr Chemoreceptor Interacts With IL-8 Provoking E. coli Transmigration Across Human Lung Epithelial Cells

    PubMed Central

    Han, Bing; Li, Manshu; Xu, Yonghao; Islam, Diana; Khang, Julie; Del Sorbo, Lorenzo; Lee, Warren; Szaszi, Katalin; Zhong, Nanshan; Slutsky, Arthur S.; Li, Yimin; Zhang, Haibo

    2016-01-01

    Bacterial colonization of epithelial surfaces and subsequent transmigration across the mucosal barrier are essential for the development of infection. We hypothesized that the methyl-accepting proteins (MCPs), known as chemoreceptors expressed on Escherichia coli (E. coli) bacterial surface, play an important role in mediating bacterial transmigration. We demonstrated a direct interaction between human interleukin-8 (IL-8) and Tsr receptor, a major MCP chemoreceptor. Stimulation of human lung epithelial cell monolayer with IL-8 resulted in increased E. coli adhesion and transmigration of the native strain (RP437) and a strain expressing only Tsr (UU2373), as compared to a strain (UU2599) with Tsr truncation. The augmented E. coli adhesion and migration was associated with a higher expression of carcinoembryonic antigen-related cell adhesion molecule 6 and production of inflammatory cytokines/chemokines, and a lower expression of the tight junction protein claudin-1 and the plasma membrane protein caveolin-1 in lung epithelial cells. An increased E. coli colonization and pulmonary cytokine production induced by the RP437 and UU2373 strains was attenuated in mice challenged with the UU2599 strain. Our results suggest a critical role of the E. coli Tsr chemoreceptor in mediating bacterial colonization and transmigration across human lung epithelium during development of pulmonary infections. PMID:27506372

  4. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    SciTech Connect

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J.

    1995-12-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.

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

    PubMed Central

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

    2015-01-01

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

  6. Alpha2 adrenoceptors regulate proliferation of human intestinal epithelial cells

    PubMed Central

    Schaak, S; Cussac, D; Cayla, C; Devedjian, J; Guyot, R; Paris, H; Denis, C

    2000-01-01

    BACKGROUND AND AIMS—Previous studies on rodents have suggested that catecholamines stimulate proliferation of the intestinal epithelium through activation of α2 adrenoceptors located on crypt cells. The occurrence of this effect awaits demonstration in humans and the molecular mechanisms involved have not yet been elucidated. Here, we examined the effect of α2 agonists on a clone of Caco2 cells expressing the human α2A adrenoceptor.
METHODS—Cells were transfected with a bicistronic plasmid containing the α2C10 and neomycin phosphotransferase genes. G418 resistant clones were assayed for receptor expression using radioligand binding. Receptor functionality was assessed by testing its ability to couple Gi proteins and to inhibit cAMP production. Mitogen activated protein kinase (MAPK) phosphorylation was followed by western blot, and cell proliferation was estimated by measuring protein and DNA content.
RESULTS—Permanent transfection of Caco2 cells allowed us to obtain a clone (Caco2-3B) expressing α2A adrenoceptors at a density similar to that found in normal human intestinal epithelium. Caco2-3B retained morphological features and brush border enzyme expression characteristic of enterocytic differentiation. The receptor was coupled to Gi2/Gi3 proteins and its stimulation caused marked diminution of forskolin induced cAMP production. Treatment of Caco2-3B with UK14304 (α2 agonist) induced a rapid increase in the phosphorylation state of MAPK, extracellular regulated protein kinase 1 (Erk1), and 2 (Erk2). This event was totally abolished in pertussis toxin treated cells and in the presence of kinase inhibitors (genistein or PD98059). It was unaffected by protein kinase C downregulation but correlated with a transient increase in Shc tyrosine phosphorylation. Finally, sustained exposure of Caco2-3B to UK14304 resulted in modest but significant acceleration of cell proliferation. None of these effects was observed in the parental cell line Caco2.

  7. 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.

  8. 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...

  9. 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.

  10. Interleukin-13 interferes with CFTR and AQP5 expression and localization during human airway epithelial cell differentiation

    SciTech Connect

    Skowron-zwarg, Marie; Boland, Sonja; Caruso, Nathalie; Coraux, Christelle; Marano, Francelyne; Tournier, Frederic . E-mail: f-tournier@paris7.jussieu.fr

    2007-07-15

    Interleukin-13 (IL-13) is a central regulator of Th2-dominated respiratory disorders such as asthma. Lesions of the airway epithelial barrier frequently observed in chronic respiratory inflammatory diseases are repaired through proliferation, migration and differentiation of epithelial cells. Our work is focused on the effects of IL-13 in human cellular models of airway epithelial cell regeneration. We have previously shown that IL-13 altered epithelial cell polarity during mucociliary differentiation of human nasal epithelial cells. In particular, the cytokine inhibited ezrin expression and interfered with its apical localization during epithelial cell differentiation in vitro. Here we show that CFTR expression is enhanced in the presence of the cytokine, that two additional CFTR protein isoforms are expressed in IL-13-treated cells and that part of the protein is retained within the endoplasmic reticulum. We further show that aquaporin 5 expression, a water channel localized within the apical membrane of epithelial cells, is completely abolished in the presence of the cytokine. These results show that IL-13 interferes with ion and water channel expression and localization during epithelial regeneration and may thereby influence mucus composition and hydration.

  11. Effect of topical microbicides on infectious human immunodeficiency virus type 1 binding to epithelial cells.

    PubMed

    Roth, Susan; Monsour, Michael; Dowland, Amanda; Guenthner, Patricia C; Hancock, Kelly; Ou, Chin-Yi; Dezzutti, Charlene S

    2007-06-01

    Topical microbicides (cellulose acetate 1,2 benzene dicarboxylate [CAP], PRO 2000, SPL7013, and UC781) are being investigated to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1). These products were shown to prevent the transfer of infectious HIV-1 from urogenital and colorectal epithelial cell lines to peripheral blood mononuclear cells. However, it was unclear if the topical microbicides rendered the virus noninfectious and/or reduced the binding to the epithelial cells. To test this, epithelial cells were cultured with HIV-1 in the presence or absence of topical microbicides or their placebos. The cells were washed, RNA lysates were made, and real-time PCR was performed for HIV-1. PRO 2000 and SPL7013 significantly (P < 0.0001) reduced the amount of bound HIV-1 to the colorectal epithelial cell line across clades A, B, C, and CRF01-AE. While none of the products reduced the binding of HIV-1 clades A and C to the urogenital cell line, CAP, PRO 2000, and SPL7013 significantly (P epithelial cells and possible shedding into mucosal secretions. Therefore, functional virological assays in addition to measuring viral RNA should be included when clinically evaluating topical microbicide use by infected persons.

  12. Effect of Topical Microbicides on Infectious Human Immunodeficiency Virus Type 1 Binding to Epithelial Cells▿

    PubMed Central

    Roth, Susan; Monsour, Michael; Dowland, Amanda; Guenthner, Patricia C.; Hancock, Kelly; Ou, Chin-Yi; Dezzutti, Charlene S.

    2007-01-01

    Topical microbicides (cellulose acetate 1,2 benzene dicarboxylate [CAP], PRO 2000, SPL7013, and UC781) are being investigated to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1). These products were shown to prevent the transfer of infectious HIV-1 from urogenital and colorectal epithelial cell lines to peripheral blood mononuclear cells. However, it was unclear if the topical microbicides rendered the virus noninfectious and/or reduced the binding to the epithelial cells. To test this, epithelial cells were cultured with HIV-1 in the presence or absence of topical microbicides or their placebos. The cells were washed, RNA lysates were made, and real-time PCR was performed for HIV-1. PRO 2000 and SPL7013 significantly (P < 0.0001) reduced the amount of bound HIV-1 to the colorectal epithelial cell line across clades A, B, C, and CRF01-AE. While none of the products reduced the binding of HIV-1 clades A and C to the urogenital cell line, CAP, PRO 2000, and SPL7013 significantly (P ≤ 0.002) reduced the binding of clades B and CRF01-AE. In general, PRO 2000 and SPL7013 placebos significantly (P < 0.0001) reduced the amount of bound HIV-1 but were less than the active products. UC781, its placebo, and hydroxyethyl cellulose (placebo for CAP) minimally affected the amount of bound HIV-1. These results suggest that rendering HIV-1 noninfectious may not correlate to the amount of HIV-1 bound to epithelial cells and possible shedding into mucosal secretions. Therefore, functional virological assays in addition to measuring viral RNA should be included when clinically evaluating topical microbicide use by infected persons. PMID:17404008

  13. Effect of Hangeshashinto on calprotectin expression in human oral epithelial cells.

    PubMed

    Hiroshima, Yuka; Bando, Mika; Inagaki, Yuji; Kido, Reiko; Kataoka, Masatoshi; Nagata, Toshihiko; Kido, Jun-Ichi

    2016-05-01

    Oral epithelial cells produce antimicrobial peptides (AMPs) to prevent microbial infection. Calprotectin (S100A8/S100A9) is one of these AMPs in oral epithelial cells, the expression of which is up-regulated by interleukin-1α (IL-1α). Hangeshashinto (HST) is a traditional Japanese herbal medicine that has anti-inflammatory effects. The purpose of this study was to investigate the effect of HST on the expression of calprotectin through the regulation of IL-1α in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with HST in the presence or absence of anti-IL-1α antibody or IL-1 receptor antagonist, or with six major components of HST (3,4-dihydroxybenzaldehyde, baicalin, ginsenoside Rb1, glycyrrhizin, oleanolic acid and berberine). The expression of S100A8, S100A9, other AMPs and cytokine mRNAs was examined by RT-PCR and quantitative real-time PCR. Calprotectin expression and IL-1α secretion were investigated by ELISA. HST (6 μg/ml) increased the expression of S100A8/S100A9 mRNAs and calprotectin protein, and also up-regulated β-defensin 2 (DEFB4) and S100A7 expression. The expression of IL-1α mRNA and its protein was slightly but significantly increased by HST. A neutralizing antibody against IL-1α and IL-1 receptor antagonist inhibited HST-up-regulated S100A8/S100A9 mRNA expression. Although 3,4-dihydroxybenzaldehyde, baicalin and ginsenoside Rb1 as HST components increased S100A8/S100A9 expression, oleanolic acid and berberine decreased their expression. These results suggest that HST increases the expression of calprotectin, DEFB4 and S100A7 in oral epithelial cells. In response to HST, up-regulation of calprotectin expression may be partially induced via IL-1α.

  14. Comparison of methods for the isolation of human breast epithelial and myoepithelial cells

    PubMed Central

    Zubeldia-Plazaola, Arantzazu; Ametller, Elisabet; Mancino, Mario; Prats de Puig, Miquel; López-Plana, Anna; Guzman, Flavia; Vinyals, Laia; Pastor-Arroyo, Eva M.; Almendro, Vanessa; Fuster, Gemma; Gascón, Pedro

    2015-01-01

    Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer. PMID

  15. 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

  16. Value of human amniotic epithelial cells in tissue engineering for cornea.

    PubMed

    Fatimah, Simat Siti; Ng, Sook Luan; Chua, Kien Hui; Hayati, Abdul Rahman; Tan, Ay Eeng; Tan, Geok Chin

    2010-11-01

    Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.

  17. A synthetic chloride channel restores chloride conductance in human cystic fibrosis epithelial cells.

    PubMed

    Shen, Bing; Li, Xiang; Wang, Fei; Yao, Xiaoqiang; Yang, Dan

    2012-01-01

    Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl(-)) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl(-) transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl(-) channels to mediate Cl(-) transport across lipid bilayer membranes is capable of restoring Cl(-) permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl(-) channel dysfunction.

  18. A Synthetic Chloride Channel Restores Chloride Conductance in Human Cystic Fibrosis Epithelial Cells

    PubMed Central

    Wang, Fei; Yao, Xiaoqiang; Yang, Dan

    2012-01-01

    Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl−) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl− transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl− channels to mediate Cl− transport across lipid bilayer membranes is capable of restoring Cl− permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl− channel dysfunction. PMID:22514656

  19. Human gastric epithelial cells contribute to gastric immune regulation by providing retinoic acid to dendritic cells.

    PubMed

    Bimczok, D; Kao, J Y; Zhang, M; Cochrun, S; Mannon, P; Peter, S; Wilcox, C M; Mönkemüller, K E; Harris, P R; Grams, J M; Stahl, R D; Smith, P D; Smythies, L E

    2015-05-01

    Despite the high prevalence of chronic gastritis caused by Helicobacter pylori, the gastric mucosa has received little investigative attention as a unique immune environment. Here, we analyzed whether retinoic acid (RA), an important homeostatic factor in the small intestinal mucosa, also contributes to gastric immune regulation. We report that human gastric tissue contains high levels of the RA precursor molecule retinol (ROL), and that gastric epithelial cells express both RA biosynthesis genes and RA response genes, indicative of active RA biosynthesis. Moreover, primary gastric epithelial cells cultured in the presence of ROL synthesized RA in vitro and induced RA biosynthesis in co-cultured monocytes through an RA-dependent mechanism, suggesting that gastric epithelial cells may also confer the ability to generate RA on gastric dendritic cells (DCs). Indeed, DCs purified from gastric mucosa had similar levels of aldehyde dehydrogenase activity and RA biosynthesis gene expression as small intestinal DCs, although gastric DCs lacked CD103. In H. pylori-infected gastric mucosa, gastric RA biosynthesis gene expression was severely disrupted, which may lead to reduced RA signaling and thus contribute to disease progression. Collectively, our results support a critical role for RA in human gastric immune regulation.

  20. Human bronchial epithelial cells express PAR-2 with different sensitivity to thermolysin.

    PubMed

    Ubl, Joachim J; Grishina, Zoryana V; Sukhomlin, Tatiana K; Welte, Tobias; Sedehizade, Fariba; Reiser, Georg

    2002-06-01

    Protease-activated receptor-2 (PAR-2) plays a role in inflammatory reactions in airway physiology. Proteases cleaving the extracellular NH(2) terminus of receptors activate or inactivate PAR, thus possessing a therapeutic potential. Using RT-PCR and immunocytochemistry, we show PAR-2 in human airway epithelial cell lines human bronchial epithelial (HBE) and A549. Functional expression of PAR-2 was confirmed by Ca(2+) imaging studies using the receptor agonist protease trypsin. The effect was abolished by soybean trypsin inhibitor and mimicked by the specific PAR-2 peptide agonist SLIGKV. Amplitude and duration of PAR-2-elicited Ca(2+) response in HBE and A549 cells depend on concentration and time of agonist superfusion. The response is partially pertussis toxin (PTX) insensitive, abolished by the phospholipase C inhibitor U-73122, and diminished by the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Cathepsin G altered neither the resting Ca(2+) level nor PAR-2-elicited Ca(2+) response. Thermolysin, a prototypic bacterial metalloprotease, induced a dose-dependent Ca(2+) response in HBE, but not A549, cells. In both cell lines, thermolysin abolished the response to a subsequent trypsin challenge but not to SLIGKV. Thus different epithelial cell types express different PAR-2 with identical responses to physiological stimuli (trypsin, SLIGKV) but different sensitivity to modifying proteases, such as thermolysin.

  1. Osteopontin improves adhesion and migration of human primary renal cortical epithelial cells during wound healing

    PubMed Central

    Wu, Jinfeng; Wang, Zuolin

    2016-01-01

    The aim of the present study was to investigate the effect of osteopontin (OPN) on adhesion and migration in human primary renal cortical epithelial cells during wound healing and Transwell assays. MTT assay was used to examine the cell viability and western blot analysis was used to examine the expression of cytoskeletal proteins and cell adhesion molecules. The results showed that overexpression of OPN had positive effects on the viability, proliferation, adhesion and migration of the human primary renal cortical epithelial cells. In addition, the integrity of the cell membrane and cytoskeleton of the epithelial cells was negatively affected by knockdown of OPN expression. The Transwell migration and a wound healing assays performed using OPN-knockdown cells suggested that OPN had a significant impact on cell migration (P=0.0421) and wound healing (P=0.0333). Therefore, OPN may be a potential target for the therapeutic modulation of skin repair to improve the healing rate and quality of wound healing. PMID:28101213

  2. Odontoblastic inductive potential of epithelial cells derived from human deciduous dental pulp.

    PubMed

    Lee, Hye-Kyung; Park, Ji-Won; Seo, You-Mi; Kim, Ha Hoon; Lee, Gene; Bae, Hyun-Sook; Park, Joo-Cheol

    2016-06-01

    For the dentin regeneration, dental epithelial cells are indispensible and must possess odontoblastic induction capability. Epithelial cell-like stem cells were recently identified in human deciduous dental pulp (DPESCs). However, their cellular characteristics remain poorly defined. The purpose of this study was to characterize DPESCs compared to HAT-7 ameloblastic cells. Expression levels of ameloblast-specific markers [odontogenic ameloblast-associated protein (Odam), matrix metalloproteinase (Mmp)-20, amelogenin, and ameloblastin] were detected in DPESCs. Co-culturing odontoblastic MDPC-23 cells with DPESCs increased expression of odontoblast differentiation markers (Dmp1 and Dspp) from days 4 to 10, while the expression of bone sialoprotein rapidly decreased. MDPC-23 cells cultured in DPESC-conditioned medium (CM) showed increased Dspp promoter activity compared with control MDPC-23 cultures. Mineralization was first observed in the CM groups from day 4 and proceeded rapidly until day 14, whereas mineralized nodules were found from day 7 in control media-cultured cells. In conclusion, DPESCs in human deciduous pulp possess ameloblast-like characteristics and differentiation properties, and substances derived from DPESCs promote odontoblastic differentiation. Thus, our results indicate that DPESCs can be a realistic epithelial source for use in odontoblastic induction and dentin formation of dental mesenchymal cells.

  3. Nanoemulsion-based mucosal adjuvant induces apoptosis in human epithelial cells

    PubMed Central

    Orzechowska, Beata U.; Kukowska-Latallo, Jolanta F.; Coulter, Alexa D.; Szabo, Zsuzsanna; Gamian, Andrzej; Myc, Andrzej

    2015-01-01

    Nanoemulsions (NEs) are adjuvants that enhance antigen penetration of the nasal mucosa, increase cellular uptake of antigens by both epithelial and dendritic cells, and promote the migration of antigen-loaded dendritic cells to regional lymph nodes within 24-hours of vaccine administration. The objective of this study was to elucidate cell death caused by W805EC NE and identify caspases and genes associated with death pathways. Consistent with this aim, we show that exposure of human epithelial cells (EC), both RPMI 2650 and FaDu, to NE results in the activation of caspases (1, 3/7, 6, 8, and 9) and the expression of genes involved in apoptotic as well as authophagy and necrosis pathways. Interestingly, the NE activates caspase 8 which promotes “immunogenic apoptosis”. The rescue assay was employed to investigate the fate of RPMI 2650 cells treated with W805EC NE. After four hour treatment with as little as 0.03% of NE no cells were rescued at 72 hours. Remarkably, immediately after four-hour treatment, the cells morphologically resembled untreated cells and most of the cells were alive. Altogether, these results suggest that NE induces death of human ECs through multiple pathways. Epithelial cell death caused by W805EC may have further implications on antigen uptake, processing, and presentation by DC's. PMID:25817825

  4. Vasoactive intestinal peptide (VIP) induces malignant transformation of the human prostate epithelial cell line RWPE-1.

    PubMed

    Fernández-Martínez, Ana B; Bajo, Ana M; Isabel Arenas, M; Sánchez-Chapado, Manuel; Prieto, Juan C; Carmena, María J

    2010-12-18

    The carcinogenic potential of vasoactive intestinal peptide (VIP) was analyzed in non-tumor human prostate epithelial cells (RWPE-1) and in vivo xenografts. VIP induced morphological changes and a migratory phenotype consistent with stimulation of expression/activity of metalloproteinases MMP-2 and MMP-9, decreased E-cadherin-mediated cell-cell adhesion, and increased cell motility. VIP increased cyclin D1 expression and cell proliferation that was blocked after VPAC(1)-receptor siRNA transfection. Similar effects were seen in RWPE-1 tumors developed by subcutaneous injection of VIP-treated cells in athymic nude mice. VIP acts as a cytokine in RWPE-1 cell transformation conceivably through epithelial-mesenchymal transition (EMT), reinforcing VIP role in prostate tumorigenesis.

  5. Pulmonary surfactant mitigates silver nanoparticle toxicity in human alveolar type-I-like epithelial cells.

    PubMed

    Sweeney, Sinbad; Leo, Bey Fen; Chen, Shu; Abraham-Thomas, Nisha; Thorley, Andrew J; Gow, Andrew; Schwander, Stephan; Zhang, Junfeng Jim; Shaffer, Milo S P; Chung, Kian Fan; Ryan, Mary P; Porter, Alexandra E; Tetley, Teresa D

    2016-09-01

    Accompanying increased commercial applications and production of silver nanomaterials is an increased probability of human exposure, with inhalation a key route. Nanomaterials that deposit in the pulmonary alveolar region following inhalation will interact firstly with pulmonary surfactant before they interact with the alveolar epithelium. It is therefore critical to understand the effects of human pulmonary surfactant when evaluating the inhalation toxicity of silver nanoparticles. In this study, we evaluated the toxicity of AgNPs on human alveolar type-I-like epithelial (TT1) cells in the absence and presence of Curosurf(®) (a natural pulmonary surfactant substitute), hypothesising that the pulmonary surfactant would act to modify toxicity. We demonstrated that 20nm citrate-capped AgNPs induce toxicity in human alveolar type I-like epithelial cells and, in agreement with our hypothesis, that pulmonary surfactant acts to mitigate this toxicity, possibly through reducing AgNP dissolution into cytotoxic Ag(+) ions. For example, IL-6 and IL-8 release by TT1 cells significantly increased 10.7- and 35-fold, respectively (P<0.01), 24h after treatment with 25μg/ml AgNPs. In contrast, following pre-incubation of AgNPs with Curosurf(®), this effect was almost completely abolished. We further determined that the mechanism of this toxicity is likely associated with Ag(+) ion release and lysosomal disruption, but not with increased reactive oxygen species generation. This study provides a critical understanding of the toxicity of AgNPs in target human alveolar type-I-like epithelial cells and the role of pulmonary surfactant in mitigating this toxicity. The observations reported have important implications for the manufacture and application of AgNPs, in particular for applications involving use of aerosolised AgNPs.

  6. 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.

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

    PubMed Central

    Ozbun, Michelle A.; Patterson, Nicole A.

    2014-01-01

    Papillomaviruses have a strict tropism for epithelial cells and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro wherein virion morphogenesis occurs under cooperative viral and cellular cues requires the cultivation of epithelium. Presented in the first section of this unit is a protocol for growing differentiating epithelial tissues, whose structure and function mimics many important morphological and biochemical aspects of normal skin. The technique, pioneered by Asslineau and Pruniéras (Asselineau and Prunieras 1984) and modified by Kopan et al. (Kopan et al. 1987), involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname “raft” cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, as well as keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single step virus growth

  8. Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.

    PubMed

    Liu, Bo; Yu, Zhuoteng; Chen, Ceng; Kling, David E; Newburg, David S

    2012-08-01

    Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate-labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

  9. 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.

  10. 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.

  11. Oxidative stress-induced epigenetic changes associated with malignant transformation of human kidney epithelial cells.

    PubMed

    Mahalingaiah, Prathap Kumar S; Ponnusamy, Logeswari; Singh, Kamaleshwar P

    2016-09-17

    Renal Cell Carcinoma (RCC) in humans is positively influenced by oxidative stress status in kidneys. We recently reported that adaptive response to low level of chronic oxidative stress induces malignant transformation of immortalized human renal tubular epithelial cells. Epigenetic alterations in human RCC are well documented, but its role in oxidative stress-induced malignant transformation of kidney cells is not known. Therefore, the objective of this study was to evaluate the potential role of epigenetic changes in chronic oxidative stress-induced malignant transformation of HK-2, human renal tubular epithelial cells. The results revealed aberrant expression of epigenetic regulatory genes involved in DNA methylation (DNMT1, DNMT3a and MBD4) and histone modifications (HDAC1, HMT1 and HAT1) in HK-2 cells malignantly transformed by chronic oxidative stress. Additionally, both in vitro soft agar assay and in vivo nude mice study showing decreased tumorigenic potential of malignantly transformed HK-2 cells following treatment with DNA de-methylating agent 5-aza 2' dC further confirmed the crucial role of DNA hypermethyaltion in oxidative stress-induced malignant transformation. Changes observed in global histone H3 acetylation (H3K9, H3K18, H3K27 and H3K14) and decrease in phospho-H2AX (Ser139) also suggest potential role of histone modifications in increased survival and malignant transformation of HK-2 cells by oxidative stress. In summary, the results of this study suggest that epigenetic reprogramming induced by low levels of oxidative stress act as driver for malignant transformation of kidney epithelial cells. Findings of this study are highly relevant in potential clinical application of epigenetic-based therapeutics for treatments of kidney cancers.

  12. Alteration of transcriptional profile in human bronchial epithelial cells induced by cigarette smoke condensate.

    PubMed

    Hu, Ying-Chun; Yang, Zhi-Hua; Zhong, Ke-Jun; Niu, Li-Jing; Pan, Xiu-Jie; Wu, De-Chang; Sun, Xian-Jun; Zhou, Ping-Kun; Zhu, Mao-Xiang; Huo, Yan-Ying

    2009-10-08

    Despite the significance of cigarette smoke for carcinogenesis, the molecular mechanisms that lead to increased susceptibility of human cancers are not well-understood. In our present study, the oncogenic transforming effects of cigarette smoke condensate (CSC) were examined using papillomavirus-immortalized human bronchial epithelial cells (BEP2D). Growth kinetics, saturation density, resistance to serum-induced terminal differentiation, anchorage-independent growth and tumorigenicity in nude mice were used to investigate the various stages of transformation in BEP2D cells. Illumina microarray platforms were used to explore the CSC-induced alteration of global mRNA expression profiles of the earlier period and the advanced stage of CSC-treated BEP2D cells. We showed here that a series of sequential steps arose among CSC-treated immortalized human bronchial epithelial cells, including altered growth kinetics, resistance to serum-induced terminal differentiation, and anchorage-independence growth. In the earlier period of CSC treatment, 265 genes were down-regulated and 63 genes were up-regulated, respectively, and in the advanced stage of CSC treatment, 313 genes were down-regulated and 145 genes were up-regulated, respectively. Notably, among those genes, the expression of some of imprinted genes such as IGF2, NDN, H19 and MEG3 were all silenced or down-regulated in CSC-treated cells. These genes reactivated after 5 microM 5-aza-2-deoxycytidine (5-aza-dC) treatment. These results demonstrated that long-term treatment of human bronchial epithelial cells with CSC may adversely affect their genetic and epigenetic integrity and lead to further transformation.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26297489

  14. Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion

    PubMed Central

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

    2015-01-01

    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. PMID:26629549

  15. Regenerative potential of human airway stem cells in lung epithelial engineering.

    PubMed

    Gilpin, Sarah E; Charest, Jonathan M; Ren, Xi; Tapias, Luis F; Wu, Tong; Evangelista-Leite, Daniele; Mathisen, Douglas J; Ott, Harald C

    2016-11-01

    Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5(+)TP63(+) basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair.

  16. 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 in cervical carcinoma in vivo.

  17. Sulfation of chlorotyrosine and nitrotyrosine by human lung endothelial and epithelial cells: Role of the human SULT1A3

    SciTech Connect

    Yasuda, Shin; Yasuda, Tomoko; Liu, Ming-Yih; Shetty, Sreerama; Idell, Steven; Boggaram, Vijayakumar; Suiko, Masahito; Sakakibara, Yoichi; Fu Jian; Liu, Ming-Cheh

    2011-03-01

    During inflammation, potent reactive oxidants formed may cause chlorination and nitration of both free and protein-bound tyrosine. In addition to serving as biomarkers of inflammation-mediated oxidative stress, elevated levels of chlorotyrosine and nitrotyrosine have been linked to the pathogenesis of lung and vascular disorders. The current study was designed to investigate whether the lung cells are equipped with mechanisms for counteracting these tyrosine derivatives. By metabolic labeling, chlorotyrosine O-[{sup 35}S]sulfate and nitrotyrosine O-[{sup 35}S]sulfate were found to be generated and released into the labeling media of human lung endothelial and epithelial cells labeled with [{sup 35}S]sulfate in the presence of added chlorotyrosine and nitrotyrosine. Enzymatic assays using the eleven known human cytosolic sulfotransferases (SULTs) revealed SULT1A3 as the enzyme responsible for catalyzing the sulfation of chlorotyrosine and nitrotyrosine. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the expression of SULT1A3 in the lung endothelial and epithelial cells used in this study. Kinetic constants of the sulfation of chlorotyrosine and nitrotyrosine by SULT1A3 were determined. Collectively, these results suggest that sulfation by SULT1A3 in lung endothelial and epithelial cells may play a role in the inactivation and/or disposal of excess chlorotyrosine and nitrotyrosine generated during inflammation.

  18. 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

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

    SciTech Connect

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

    2007-07-06

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

  20. 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.

  1. Barrier-protective effects of activated protein C in human alveolar epithelial cells.

    PubMed

    Puig, Ferranda; Fuster, Gemma; Adda, Mélanie; Blanch, Lluís; Farre, Ramon; Navajas, Daniel; Artigas, Antonio

    2013-01-01

    Acute lung injury (ALI) is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC) on mechanical tension and barrier integrity in human alveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml) or vehicle (control). Subsequently, thrombin (50 nM) or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

  2. Neisserial Heparin Binding Antigen (NHBA) Contributes to the Adhesion of Neisseria meningitidis to Human Epithelial Cells

    PubMed Central

    Vacca, Irene; Del Tordello, Elena; Gasperini, Gianmarco; Pezzicoli, Alfredo; Di Fede, Martina; Rossi Paccani, Silvia; Marchi, Sara; Mubaiwa, Tsisti D.; Hartley-Tassell, Lauren E.; Jennings, Michael P.; Seib, Kate L.; Masignani, Vega; Pizza, Mariagrazia; Serruto, Davide; Aricò, Beatrice; Delany, Isabel

    2016-01-01

    Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and an antigen of the Bexsero® vaccine. NHBA binds heparin through a conserved Arg-rich region that is the target of two proteases, the meningococcal NalP and human lactoferrin (hLf). In this work, in vitro studies showed that recombinant NHBA protein was able to bind epithelial cells and mutations of the Arg-rich tract abrogated this binding. All N-terminal and C-terminal fragments generated by NalP or hLf cleavage, regardless of the presence or absence of the Arg-rich region, did not bind to cells, indicating that a correct positioning of the Arg-rich region within the full length protein is crucial. Moreover, binding was abolished when cells were treated with heparinase III, suggesting that this interaction is mediated by heparan sulfate proteoglycans (HSPGs). N. meningitidis nhba knockout strains showed a significant reduction in adhesion to epithelial cells with respect to isogenic wild-type strains and adhesion of the wild-type strain was inhibited by anti-NHBA antibodies in a dose-dependent manner. Overall, the results demonstrate that NHBA contributes to meningococcal adhesion to epithelial cells through binding to HSPGs and suggest a possible role of anti-Bexsero® antibodies in the prevention of colonization. PMID:27780200

  3. Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells

    SciTech Connect

    Yaswen, P.; Smoll, A.; Stampfer, M.R. ); Peehl, D.M. ); Trask, D.K.; Sager, R. )

    1990-10-01

    A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4 kilobase mRNA, designated NB-1, whose expression was decreased >50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo({alpha})pyrene and Kirsten sarcoma virus. Sequence analysis of NB-1 cDNA revealed an open reading frame with a high degree of homology to calmodulin. NB-1 expression could be demonstrated by polymerase chain reaction amplification in normal breast, prostate, cervix, and epidermal tissues. The presence of NB-1 transcripts was variable in primary breast carcinoma tissues and undetectable in tumor-derived cell lines of breast, prostate, or other origins. NB-1 mRNA expression could be down-regulated in cultured HMECs by exposure to reconstituted extracellular matrix material, while exposure to transforming growth factor type {beta} increased its relative abundance. The protein encoded by NB-1 may have Ca{sup 2{sup plus}} binding properties and perform functions similar to those of authentic calmodulin. Its possible roles in differentiation and/or suppression of tumorigenicity in epithelial tissues remain to be examined.

  4. Soluble extracellular Klotho decreases sensitivity to cigarette smoke induced cell death in human lung epithelial cells.

    PubMed

    Blake, David J; Reese, Caitlyn M; Garcia, Mario; Dahlmann, Elizabeth A; Dean, Alexander

    2015-10-01

    Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death in the US and is associated with an abnormal inflammatory response to cigarette smoke (CS). Exposure to CS induces oxidative stress and can result in cellular senescence in the lung. Cellular senescence can then lead to decreased proliferation of epithelial cells, the destruction of alveolar structure and pulmonary emphysema. The anti-aging gene, klotho, encodes a membrane bound protein that has been shown to be a key regulator of oxidative stress and cellular senescence. In this study the role of Klotho (KL) with regard to oxidative stress and cellular senescence was investigated in human pulmonary epithelial cells exposed to cigarette smoke. Individual clones that stably overexpress Klotho were generated through retroviral transfection and geneticin selection. Klotho overexpression was confirmed through RT-qPCR, Western blotting and ELISA. Compared to control cells, constitutive Klotho overexpression resulted in decreased sensitivity to cigarette smoke induced cell death in vitro via a reduction of reactive oxygen species and a decrease in the expression of p21. Our results suggest that increasing Klotho level in pulmonary epithelial cells may be a promising strategy to reduce cellular senescence and mitigate the risk for the development of COPD.

  5. Secondhand smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial cells

    PubMed Central

    2009-01-01

    Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure. PMID:19943936

  6. Human primary bronchial epithelial cells respond differently to titanium dioxide nanoparticles than the lung epithelial cell lines A549 and BEAS-2B.

    PubMed

    Ekstrand-Hammarström, Barbro; Akfur, Christine M; Andersson, Per Ola; Lejon, Christian; Osterlund, Lars; Bucht, Anders

    2012-09-01

    We have compared the cellular uptake and responses of five preparations of nanocrystalline titanium dioxide (TiO(2)) between normal human bronchial epithelial (NHBE) cells and epithelial cell lines (A549 and BEAS-2B). The P25 nanoparticles, containing both anatase and rutile modifications, induced reactive oxygen species (ROS) and secretion of the neutrophil chemoattractant IL-8 in all three cell types used. Pure anatase and rutile particles provoked differential IL-8 response in A549 and no response in BEAS-2B cells despite similar formation of ROS. The pure TiO(2) modifications also provoked release of the inflammatory mediators: IL-6, G-CSF and VEGF, in NHBE cells but not in the two cell lines. We conclude that the responsiveness of lung epithelial cells is strongly dependent on both the physicochemical properties of TiO(2) nanoparticles and the type of responder cells. The differential pro-inflammatory responsiveness of primary lung epithelial cells compared with immortalized cell lines should be considered in the assessment of adverse reactions to inhaled nanoparticles.

  7. Mesenchymal-epithelial transitions: spontaneous and cumulative syntheses of epithelial marker molecules and their assemblies to novel cell junctions connecting human hematopoietic tumor cells to carcinomatoid tissue structures.

    PubMed

    Franke, Werner W; Rickelt, Steffen

    2011-12-01

    Using biochemical as well as light- and electron-microscopic immunolocalization methods, in cultures of unicellular human blood tumor cells, we have studied the phenomenon of spontaneous and cumulative syntheses of certain epithelial proteins and glycoproteins and their assemblies to two major kinds of novel cell-cell junctions, adhering junctions (AJs) and junctions based on the epithelial cell adhesion molecule (EpCAM). More than two decades, we have selected and characterized clonal sublines of multipotential hematopoietic K562 cells, which are enriched in newly formed AJs based on cis-clusters of desmoglein Dsg2, in some sublines accompanied by desmocollin Dsc2. Both desmosomal cadherins can be anchored in a submembranous plaque containing plakoglobin and plakophilins Pkp2 and Pkp3, with or without other armadillo proteins and desmoplakin. Also, these cells are often connected by an additional, extended junction system, in which the transmembrane epithelial glycoprotein EpCAM is associated with a cytoplasmic plaque rich in several actin-binding proteins such as afadin, α-actinin, ezrin and vinculin. Both kinds of junctions contribute to connections of K562 cells into epithelioid monolayers or even three-dimensional, tissue-like structures, thus markedly changing the cell biological nature and behavior of the resulting tumor subforms (mesenchymal-epithelial transitions). We discuss molecular mechanisms involved in the formation and function of these junctions, also with respect to tumor spread and metastasis, as well as diagnostic and therapeutic consequences.

  8. In vitro ultraviolet–induced damage in human corneal, lens, and retinal pigment epithelial cells

    PubMed Central

    Youn, Hyun-Yi; Sivak, Jacob G.; Jones, Lyndon W.

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope. Results The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. Conclusions The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the

  9. Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells

    PubMed Central

    Castillo, Lilian Fedra; Tascón, Rocío; Huvelle, María Amparo Lago; Novack, Gisela; Llorens, María Candelaria; dos Santos, Ancely Ferreira; Shortrede, Jorge; Cabanillas, Ana María; Joffé, Elisa Bal de Kier; Labriola, Leticia; Peters, María Giselle

    2016-01-01

    Breast cancer is the disease with the highest impact on global health, being metastasis the main cause of death. To metastasize, carcinoma cells must reactivate a latent program called epithelial-mesenchymal transition (EMT), through which epithelial cancer cells acquire mesenchymal-like traits. Glypican-3 (GPC3), a proteoglycan involved in the regulation of proliferation and survival, has been associated with cancer. In this study we observed that the expression of GPC3 is opposite to the invasive/metastatic ability of Hs578T, MDA-MB231, ZR-75-1 and MCF-7 human breast cancer cell lines. GPC3 silencing activated growth, cell death resistance, migration, and invasive/metastatic capacity of MCF-7 cancer cells, while GPC3 overexpression inhibited these properties in MDA-MB231 tumor cell line. Moreover, silencing of GPC3 deepened the MCF-7 breast cancer cells mesenchymal characteristics, decreasing the expression of the epithelial marker E-Cadherin. On the other side, GPC3 overexpression induced the mesenchymal-epithelial transition (MET) of MDA-MB231 breast cancer cells, which re-expressed E-Cadherin and reduced the expression of vimentin and N-Cadherin. While GPC3 inhibited the canonical Wnt/β-Catenin pathway in the breast cancer cells, this inhibition did not have effect on E-Cadherin expression. We demonstrated that the transcriptional repressor of E-Cadherin - ZEB1 - is upregulated in GPC3 silenced MCF-7 cells, while it is downregulated when GPC3 was overexpressed in MDA-MB231 cells. We presented experimental evidences showing that GPC3 induces the E-Cadherin re-expression in MDA-MB231 cells through the downregulation of ZEB1. Our data indicate that GPC3 is an important regulator of EMT in breast cancer, and a potential target for procedures against breast cancer metastasis. PMID:27507057

  10. Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine

    PubMed Central

    Khazaei, Mohammad Rasoul; Rashidi, Zahra; Chobsaz, Farzaneh; Khazaei, Mozafar

    2016-01-01

    Objective(s): Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. Materials and Methods: In this in vitro study, endometrial biopsies from endometriosis patients (n=9) were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml). Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0), 10, 25, 50 and 100 micromole/liter (µM) concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO) concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO)–Ethidium Bromide (EB) double staining and Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. Data were analyzed by one-way ANOVA. Results: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P<0.05) and apoptotic index increased in 25, 50 and 100 µM noscapine concentrations in 48 hr significantly (P<0.05). Concentrations of NO didn’t show a significant decrease. Conclusion: Noscapine increased endometriotic epithelial and stromal cell death and can be suggested as a treatment for endometriosis. PMID:27803780

  11. Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells.

    PubMed

    Castillo, Lilian Fedra; Tascón, Rocío; Lago Huvelle, María Amparo; Novack, Gisela; Llorens, María Candelaria; Dos Santos, Ancely Ferreira; Shortrede, Jorge; Cabanillas, Ana María; Bal de Kier Joffé, Elisa; Labriola, Leticia; Peters, María Giselle

    2016-09-13

    Breast cancer is the disease with the highest impact on global health, being metastasis the main cause of death. To metastasize, carcinoma cells must reactivate a latent program called epithelial-mesenchymal transition (EMT), through which epithelial cancer cells acquire mesenchymal-like traits.Glypican-3 (GPC3), a proteoglycan involved in the regulation of proliferation and survival, has been associated with cancer. In this study we observed that the expression of GPC3 is opposite to the invasive/metastatic ability of Hs578T, MDA-MB231, ZR-75-1 and MCF-7 human breast cancer cell lines. GPC3 silencing activated growth, cell death resistance, migration, and invasive/metastatic capacity of MCF-7 cancer cells, while GPC3 overexpression inhibited these properties in MDA-MB231 tumor cell line. Moreover, silencing of GPC3 deepened the MCF-7 breast cancer cells mesenchymal characteristics, decreasing the expression of the epithelial marker E-Cadherin. On the other side, GPC3 overexpression induced the mesenchymal-epithelial transition (MET) of MDA-MB231 breast cancer cells, which re-expressed E-Cadherin and reduced the expression of vimentin and N-Cadherin. While GPC3 inhibited the canonical Wnt/β-Catenin pathway in the breast cancer cells, this inhibition did not have effect on E-Cadherin expression. We demonstrated that the transcriptional repressor of E-Cadherin - ZEB1 - is upregulated in GPC3 silenced MCF-7 cells, while it is downregulated when GPC3 was overexpressed in MDA-MB231 cells. We presented experimental evidences showing that GPC3 induces the E-Cadherin re-expression in MDA-MB231 cells through the downregulation of ZEB1.Our data indicate that GPC3 is an important regulator of EMT in breast cancer, and a potential target for procedures against breast cancer metastasis.

  12. Sprouty2 Suppresses Epithelial-Mesenchymal Transition of Human Lens Epithelial Cells through Blockade of Smad2 and ERK1/2 Pathways

    PubMed Central

    Chen, Chuan; Chen, Xiaoyun; Qin, Yingyan; Qu, Bo; Luo, Lixia; Lin, Haotian; Wu, Mingxing; Chen, Weirong; Liu, Yizhi

    2016-01-01

    Transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) plays a key role in the pathogenesis of anterior subcapsular cataract (ASC) and capsule opacification. In mouse lens, Sprouty2 (Spry2) has a negative regulatory role on TGFβ signaling. However, the regulation of Spry2 during ASC development and how Spry2 modulates TGFβ signaling pathway in human LECs have not been characterized. Here, we demonstrate that Spry2 expression level is decreased in anterior capsule LECs of ASC patients. Spry2 negatively regulates TGFβ2-induced EMT and migration of LECs through inhibition of Smad2 and ERK1/2 phosphorylation. Also, blockade of Smad2 or ERK1/2 activation suppresses EMT caused by Spry2 downregulation. Collectively, our results for the first time show in human LECs that Spry2 has an inhibitory role in TGFβ signaling pathway. Our findings in human lens tissue and epithelial cells suggest that Spry2 may become a novel therapeutic target for the prevention and treatment of ASC and capsule opacification. PMID:27415760

  13. 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).

  14. Lycium barbarum Polysaccharides Protect Human Lens Epithelial Cells against Oxidative Stress–Induced Apoptosis and Senescence

    PubMed Central

    Wen, Yuechun; Liu, Lian; Guo, Xiaoling; Hou, Guanghui; Wang, Guifang; Zhong, Jingxiang

    2014-01-01

    Objectives We aimed to investigate the protective effect of Lycium barbarum polysaccharides (LBPs) against oxidative stress–induced apoptosis and senescence in human lens epithelial cells. Methods To study apoptosis, SRA01/04 cells, a human lens epithelial cell lines, were exposed to 200 µM hydrogen peroxide (H2O2) for 24 h with or without pretreatment with LBPs. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, intracellular reactive oxygen species (ROS), and the loss of mitochondria membrane potential (Δψm) were detected by flow cytometric analyses. Expression levels of Bcl-2 and Bax proteins were measured by western blot analysis. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were quantized using commercial enzymatic kits according to the manufacturer's instructions. To study senescence, SRA01/04 cells were pre-incubated with LBPs and all cells were then exposed to 100 µM H2O2 for 96 h. Cellular senescence was assessed by morphologic examination and senescence-associated β-galactosidase (SA-β-gal) staining. Results LBPs significantly reduced H2O2-induced cell apoptosis, the generation of ROS, the loss of Δψm, and the levels of MDA. LBPs also inhibited H2O2-induced downregulated Bcl-2 and upregulated Bax proteins and increased the levels of SOD and GSH enzyme activity. Moreover, LBPs significantly attenuated H2O2-induced cellular senescence. Conclusions These findings suggested that LBPs protect human lens epithelial cells from H2O2-induced apoptosis by modulating the generation of ROS, loss of Δψm, Bcl-2 family, and antioxidant enzyme activity and attenuating cellular senescence. PMID:25333784

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

    PubMed

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

    1995-03-01

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

  16. Induction of human alveolar epithelial cell growth factor receptors by dendrimeric nanostructures.

    PubMed

    Omidi, Yadollah; Barar, Jaleh

    2009-01-01

    Although nonviral dendrimeric nanostructures have been widely used as gene delivery systems, key questions about target cells responses to these nanostructures are yet to be answered. Here, we report the responsiveness of A431 and A549 cells upon treatment with polypropylenimine diaminobutane (DAB) dendrimers nanosystems. Complexation of DAB dendrimers with DNA reduced the zeta potential of nanostructures, but increased their size. Fluorescence microscopy revealed high transfection efficiency in both cell lines treated with DAB dendrimers with induced cytotoxicity evidenced by MTT assay. The A549 cells showed upregulation of epidermal growth factor receptor (EGFR) and its downstream signalling biomolecule Akt kinase upon treatment with DAB dendrimers, while no changes were observed in A431 cells. Based on our findings, the biological impacts of these nanosystems appeared to be cell dependent. Thus, the biological responses of target cells should be taken into account when these nanostructures are used as gene delivery system.

  17. Human oral mucosal epithelial cell sheets imaging with high-resolution phase-diversity homodyne OCT

    NASA Astrophysics Data System (ADS)

    Senda, Naoko; Osawa, Kentaro

    2015-03-01

    There is a need for development of non-invasive technique to evaluate regenerative tissues such as cell sheets for transplantation. We demonstrated non-invasive imaging inside living cell sheets of human oral mucosal epithelial cells by phase-diversity homodyne optical coherence tomography (OCT). The new method OCT developed in Hitachi enables cell imaging because of high resolution (axial resolution; ~2.6 μm, lateral resolution; ~1 μm, in the air). Nuclei inside cell sheets were imaged with sufficient spatial resolution to identify each cell. It suggested that the new method OCT could be useful for non-invasive cell sheet evaluation test.

  18. Grepafloxacin inhibits tumor necrosis factor-alpha-induced interleukin-8 expression in human airway epithelial cells.

    PubMed

    Hashimoto, S; Matsumoto, K; Gon, Y; Maruoka, S; Hayashi, S; Asai, Y; Machino, T; Horie, T

    2000-01-01

    We examined the effect of grepafloxacin (GPFX), a new fluoroquinolone antimicrobial agent, on interleukin-8 (IL-8) expression in tumor necrosis factor-alpha (TNF-alpha)-stimulated human airway epithelial cells (AEC). GPFX inhibited IL-8 protein production as well as mRNA expression in a concentration-dependent manner (2.5 - 25 micro g/ml), but the inhibition of IL-8 expression by corresponding concentrations of GPFX to serum and airway lining fluids was not complete. We discuss the modulatory effect of GPFX on IL-8 production in the context of its efficacy on controlling chronic airway inflammatory diseases.

  19. 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.

  20. The V protein of canine distemper virus is required for virus replication in human epithelial cells.

    PubMed

    Otsuki, Noriyuki; Nakatsu, Yuichiro; Kubota, Toru; Sekizuka, Tsuyoshi; Seki, Fumio; Sakai, Kouji; Kuroda, Makoto; Yamaguchi, Ryoji; Takeda, Makoto

    2013-01-01

    Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

  1. BAX gene over-expression via nucleofection to induce apoptosis in human lens epithelial cells.

    PubMed

    Fang, Yanwen; Mo, Xiaofen; Luo, Yi; Lu, Yi

    2012-09-01

    Despite significant advances in cataract surgery techniques, posterior capsule opacification (PCO) remains a common complication. In PCO, remaining epithelial cells cloud the lens capsule and impair postoperative vision. This in vitro study was designed to investigate the potential of a gene-based approach, specifically over-expression of the proapoptotic BAX gene, to prevent PCO. Human lens epithelial cells (HLECs) were transfected by nucleofection with a plasmid encoding a fusion protein of green fluorescent protein and human BAX. The expression levels of BAX and its antiapoptotic counterpart BCL2 were determined by realtime reverse transcription polymerase chain reaction, Western blotting and immunofluorescence. BAX over-expression-induced cell death was analyzed by fluorescence-activated cell sorting using the Annexin V antibody. Fluorescence microscopy and transmission electron microscopy were used to assess changes in morphology and ultrastructure. Differential expression of the downstream apoptosis-related factor, caspase 3, was detected by Western blotting. Nucleofection efficiency was high (nearly 80%). BAX-transfected HLECs showed remarkably enhanced BAX gene expression and BAX:BCL2 ratio, but relatively little change in endogenous BCL2 expression. BAX over-expression also led to significant cytotoxicity, induction of apoptosis-related characteristics and activation of caspase 3. In conclusion, our results indicate that BAX gene over-expression can trigger cell death in HLECs via an apoptotic pathway. Thus, BAX may be a promising candidate for human gene therapy to treat PCO.

  2. 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.

  3. Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms

    PubMed Central

    Wörmann, Mirka E.; Horien, Corey L.; Johnson, Errin; Liu, Guangyu; Aho, Ellen; Tang, Christoph M.

    2016-01-01

    In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host–pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus–pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms. PMID:26813911

  4. Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms.

    PubMed

    Wörmann, Mirka E; Horien, Corey L; Johnson, Errin; Liu, Guangyu; Aho, Ellen; Tang, Christoph M; Exley, Rachel M

    2016-03-01

    In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

  5. Fibulin-5 localisation in human endometrial cancer shifts from epithelial to stromal with increasing tumour grade, and silencing promotes endometrial epithelial cancer cell proliferation

    PubMed Central

    WINSHIP, AMY LOUISE; RAINCZUK, KATE; TON, AMANDA; DIMITRIADIS, EVA

    2016-01-01

    Endometrial cancer is the most common invasive gynaecological malignancy. While endocrine, genetic and inflammatory factors are thought to contribute to its pathogenesis, its precise etiology and molecular regulators remain poorly understood. Fibulin-5 is an extracellular matrix (ECM) protein that inhibits cell growth and invasion in several cancer cell types and is downregulated in a number of types of human cancer. However, it is unknown whether fibulin-5 plays a role in endometrial tumourigenesis. In the current report, the expression and localisation of fibulin-5 in type I endometrioid human endometrial cancers of grades (G) 1–3 was investigated using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Fibulin-5 mRNA was found to be significantly reduced in whole tumour tissues from women across G1-3 compared with benign endometrium (P<0.0001). Consistently, fibulin-5 protein was also reduced in the tumour epithelial compartment across increasing tumour grades. By contrast, increased protein localisation to the tumour stroma was observed with increasing grade. Knockdown by small interfering RNA in Ishikawa endometrial epithelial cancer cells expressing fibulin-5 stimulated cell adhesion and proliferation in vitro. Fibulin-5 mRNA expression in Ishikawa cells was induced by transforming growth factor-β and fibulin-5 in turn activated extracellular signal-regulated kinases (ERK1/2), suggesting that it may act via the mitogen-activated protein kinase pathway. In summary, the present study identified fibulin-5 as a downregulated ECM gene in human endometrial cancer and observed a shift from epithelial to stromal protein localisation with increasing tumour grade in women. These data suggest that loss of fibulin-5 function may promote endometrial cancer progression by enhancing epithelial cell adhesion and proliferation. PMID:27347195

  6. Respiratory epithelial cell expression of human transforming growth factor-alpha induces lung fibrosis in transgenic mice.

    PubMed Central

    Korfhagen, T R; Swantz, R J; Wert, S E; McCarty, J M; Kerlakian, C B; Glasser, S W; Whitsett, J A

    1994-01-01

    Increased production of EGF or TGF-alpha by the respiratory epithelial cells has been associated with the pathogenesis of various forms of lung injury. Growth factors and cytokines are thought to act locally, via paracrine and autocrine mechanisms, to stimulate cell proliferation and matrix deposition by interstitial lung cells resulting in pulmonary fibrosis. To test whether TGF-alpha mediates pulmonary fibrotic responses, we have generated transgenic mice expressing human TGF-alpha under control of regulatory regions of the human surfactant protein C (SP-C) gene. Human TGF-alpha mRNA was expressed in pulmonary epithelial cells in the lungs of the transgenic mice. Adult mice bearing the SP-C-TGF-alpha transgene developed severe pulmonary fibrosis. Fibrotic lesions were observed in peribronchial, peribronchiolar, and perivascular regions, as well as subjacent to pleural surfaces. Lesions consisted of fibrous tissue that included groups of epithelial cells expressing endogenous SP-C mRNA, consistent with their identification as distal respiratory epithelial cells. Peripheral fibrotic regions consisted of thickened pleura associated with extensive collagen deposition. Alveolar architecture was disrupted in the transgenic mice with loss of alveoli in the lung parenchyma. Pulmonary epithelial cell expression of TGF-alpha in transgenic mice disrupts alveolar morphogenesis and produces fibrotic lesions mediated by paracrine signaling between respiratory epithelial and interstitial cells of the lung. Images PMID:8163670

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

    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.Inhaled nanoparticles have a high deposition rate in

  8. Epithelial Expression of Human ABO Blood Group Genes Is Dependent upon a Downstream Regulatory Element Functioning through an Epithelial Cell-specific Transcription Factor, Elf5.

    PubMed

    Sano, Rie; Nakajima, Tamiko; Takahashi, Yoichiro; Kubo, Rieko; Kobayashi, Momoko; Takahashi, Keiko; Takeshita, Haruo; Ogasawara, Kenichi; Kominato, Yoshihiko

    2016-10-21

    The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells.

  9. Utility of human amniotic membrane allograft in re-epithelialization of the nasal tip

    PubMed Central

    Dennis, D'Antonio C.; Turnock, Adam R.; Sutton, Collin; Chastant, Bradley; Vanderlan, Wesley B.

    2016-01-01

    Variations in skin thickness and contours pose significant challenges to reconstruction of the lower third of the nose. Human amniotic membrane allograft offers a potential alternative to tissue transfer in reconstruction of the lower third of the nose. We reviewed the procedure and photographs of a healthy 56-year-old male with a 22 × 18 mm lower third nasal defect involving full thickness skin and subcutaneous tissue. Following preparation for grafting, dehydrated human amniotic membrane was fashioned to the dimensions of the defect and applied. No further surgical intervention was provided for 3 months. Complete re-epithelialization of the nasal and adjacent defects was achieved with minimal scar formation. Human amniotic membrane allograft provides an efficacious and cosmetically acceptable alternative to local and regional tissue transfer.

  10. Identification and functional characterization of breast cancer resistance protein in human bronchial epithelial cells (Calu-3)

    PubMed Central

    Paturi, Durga Kalyani; Kwatra, Deep; Ananthula, Hari Krishna; Pal, Dhananjay; Mitra, Ashim K.

    2010-01-01

    Breast cancer resistance protein (BCRP), a 72 kDa protein belongs to the subfamily G of the human ATP binding cassette transporter superfamily. Overexpression of BCRP was found to play a major role in the development of resistance against various chemotherapeutic agents. BCRP plays an important role in absorption, distribution and elimination of several therapeutic agents. BCRP expression and functional activity across human bronchial epithelium and its impact on pulmonary drug accumulation has not been established. The objective of this study is to identify and characterize the BCRP efflux transporter across human bronchial epithelium. Calu-3, a human bronchial epithelial cell line was employed as a model for this study. Reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunocytochemical studies were performed to identify and characterize the expression of BCRP. RT-PCR studies detected ABCG2 mRNA levels in Calu-3 cells. A strong band for BCRP with a molecular weight of approximately 72 kDa was observed in Western blot analysis. Immunocytochemical studies confirmed the presence of BCRP on the apical membrane of human bronchial epithelium. Functional activity of BCRP was determined by performing uptake of radioactive substrate [3H]-mitoxantrone in the presence and absence of BCRP inhibitors. Uptake of [3H]-mitoxantrone was elevated significantly in the presence of GF120918 and fumitremorgin C. An increase in the accumulation of Hoechst 33342, a fluorescent dye was also detected in the presence of BCRP inhibitors when compared to control. In summary, this study provides evidence for the presence of an ATP dependent, membrane bound efflux transporter BCRP across human bronchial epithelial cell line, Calu-3. PMID:19782742

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

    PubMed Central

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

    2010-01-01

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

  12. Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

    PubMed Central

    Davis, A. Sally; Chertow, Daniel S.; Moyer, Jenna E.; Suzich, Jon; Sandouk, Aline; Dorward, David W.; Logun, Carolea; Shelhamer, James H.

    2015-01-01

    Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic. PMID:25604814

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

    PubMed

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

    2009-03-01

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

  14. Androgen-Sensitized Apoptosis of HPr-1AR Human Prostate Epithelial Cells

    PubMed Central

    Chen, Congcong; Dienhart, Jason A.; Bolton, Eric C.

    2016-01-01

    Androgen receptor (AR) signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. The mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5α-dihydrotestosterone (DHT) treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS), TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP) in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2) were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1) were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins, such that

  15. 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.

  16. Human endometrial mesenchymal stem cells exhibit intrinsic anti-tumor properties on human epithelial ovarian cancer cells

    PubMed Central

    Bu, Shixia; Wang, Qian; Zhang, Qiuwan; Sun, Junyan; He, Biwei; Xiang, Charlie; Liu, Zhiwei; Lai, Dongmei

    2016-01-01

    Epithelial ovarian cancer (EOC) is the most lethal tumor of all gynecologic tumors. There is no curative therapy for EOC thus far. The tumor-homing ability of adult mesenchymal stem cells (MSCs) provide the promising potential to use them as vehicles to transport therapeutic agents to the site of tumor. Meanwhile, studies have showed the intrinsic anti-tumor properties of MSCs against various kinds of cancer, including epithelial ovarian cancer. Human endometrial mesenchymal stem cells (EnSCs) derived from menstrual blood are a novel source for adult MSCs and exert restorative function in some diseases. Whether EnSCs endow innate anti-tumor properties on EOC cells has never been reported. By using tumor-bearing animal model and ex vivo experiments, we found that EnSCs attenuated tumor growth by inducing cell cycle arrest, promoting apoptosis, disturbing mitochondria membrane potential and decreasing pro-angiogenic ability in EOC cells in vitro and/or in vivo. Furthermore, EnSCs decreased AKT phosphorylation and promoted nuclear translocation of Forkhead box O-3a (FoxO3a) in EOC cells. Collectively, our findings elucidated the potential intrinsic anti-tumor properties of EnSCs on EOC cells in vivo and in vitro. This research provides a potential strategy for EnSC-based anti-cancer therapy against epithelial ovarian cancer. PMID:27845405

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

    PubMed Central

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

    2014-01-01

    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/cm2). 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. PMID:25032206

  18. 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.

  19. Selective prostacyclin receptor agonism augments glucocorticoid-induced gene expression in human bronchial epithelial cells.

    PubMed

    Wilson, Sylvia M; Shen, Pamela; Rider, Christopher F; Traves, Suzanne L; Proud, David; Newton, Robert; Giembycz, Mark A

    2009-11-15

    Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57(kip2)) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to

  20. α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke.

    PubMed

    Berman, Reena; Jiang, Di; Wu, Qun; Chu, Hong Wei

    2016-01-01

    Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air-liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×10(4) 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection.

  1. Arsenic Exposure Transforms Human Epithelial Stem/Progenitor Cells into a Cancer Stem-like Phenotype

    PubMed Central

    Tokar, Erik J.; Diwan, Bhalchandra A.; Waalkes, Michael P.

    2010-01-01

    Background Inorganic arsenic is a ubiquitous environmental carcinogen affecting millions of people worldwide. Evolving theory predicts that normal stem cells (NSCs) are transformed into cancer stem cells (CSCs) that then drive oncogenesis. In humans, arsenic is carcinogenic in the urogenital system (UGS), including the bladder and potentially the prostate, whereas in mice arsenic induces multiorgan UGS cancers, indicating that UGS NSCs may represent targets for carcinogenic initiation. However, proof of emergence of CSCs induced by arsenic in a stem cell population is not available. Methods We continuously exposed the human prostate epithelial stem/progenitor cell line WPE-stem to an environmentally relevant level of arsenic (5 μM) in vitro and determined the acquired cancer phenotype. Results WPE-stem cells rapidly acquired a malignant CSC-like phenotype by 18 weeks of exposure, becoming highly invasive, losing contact inhibition, and hypersecreting matrix metalloproteinase-9. When hetero-transplanted, these cells (designated As-CSC) formed highly pleomorphic, aggressive tumors with immature epithelial- and mesenchymal-like cells, suggesting a highly pluripotent cell of origin. Consistent with tumor-derived CSCs, As-CSCs formed abundant free-floating spheres enriched in CSC-like cells, as confirmed by molecular analysis and the fact that only these floating cells formed xenograft tumors. An early loss of NSC self-renewal gene expression (p63, ABCG2, BMI-1, SHH, OCT-4, NOTCH-1) during arsenite exposure was subsequently reversed as the tumor suppressor gene PTEN was progressively suppressed and the CSC-like phenotype acquired. Conclusions Arsenite transforms prostate epithelial stem/progenitor cells into CSC-like cells, indicating that it can produce CSCs from a model NSC population. PMID:20056578

  2. Sirtuin 3 Protects against Urban Particulate Matter-Induced Autophagy in Human Bronchial Epithelial Cells.

    PubMed

    Chen, I-Chieh; Huang, Hsin-Hsiu; Chen, Pei-Fen; Chiang, Hung-Che

    2016-07-01

    Urban particulate matter (urban PM) is a heterogeneous mixture of various types of particles originating from different sources. Exposure to high concentrations of urban PM leading to adverse health effects is evaluated by using in vitro cultures of human lung epithelial cells. However, the mechanism underlying the correlation between high concentrations of urban PM exposure and adverse health effects has not been fully elucidated; urban PM-induced oxidative stress is considered as an important mechanism of urban PM-mediated cytotoxicity. Sirtuin 3 (SIRT3), a primary mitrochondrial deacetylase, controls cellular reactive oxygen species (ROS) production, and expression of antioxidant enzymes. In this study, we examined the role of SIRT3 in the regulation of urban PM-induced oxidative stress in normal primary human bronchial epithelial cells (HBEpiCs). Cell viability showed a time- and concentration-dependent decrease when exposed to urban PM, which could indicate that the amount of lactate dehydrogenase released from the cell in response to urban PM is related to cell viability in HBEpiC. The effects of urban PM on morphological and biochemical markers of autophagy in HBEpiC were analyzed by electron microscopy and Western blotting. Overexpression of SIRT3 inhibited urban PM-induced ROS generation, while concomitantly increasing the expression of antioxidant enzymes, and decreasing NF-κB activation and release of inflammation factors. Up-regulation of SIRT3 significantly inhibited the expression of autophagy markers and autophagic vacuole formation. Our findings provide a valuable insight into the potential role of the SIRT3 enzyme in regulating urban PM-induced autophagy by mediating urban PM-induced oxidative stress, which may contribute to urban PM-induced impairment of airway epithelial cell function.

  3. Human cytomegalovirus tropism for endothelial/epithelial cells: scientific background and clinical implications.

    PubMed

    Revello, M Grazia; Gerna, Giuseppe

    2010-05-01

    Human cytomegalovirus (HCMV) has been routinely isolated from and propagated in vitro in human embryonic lung fibroblast (HELF) cell cultures, while in vivo it is known to infect predominantly endothelial and epithelial cells. In recent years, genetic determinants of the HCMV tropism for endothelial/epithelial cells were identified in the UL131A/UL130/UL128 locus of HCMV genome of wild-type strains. UL131A-UL128 gene products form a complex with glycoprotein H (gH) and L (gL) resulting in a gH/gL/UL131A-UL128 complex that is required for HCMV entry into endothelial/epithelial cells. In contrast, virus entry into fibroblasts has its genetic determinants in the complex gH/gL/gO (or gH/gL). During primary HCMV infection, the neutralising antibody response measured in endothelial cells (EC) is potent, occurs very early and is directed mostly against combinations of two or three gene products of the UL131A-128 locus. On the contrary, neutralising antibodies measured in fibroblasts appear late, are relatively weak in potency and are directed against gH and gB. The T-cell immune response to UL131A-UL128 gene products remains to be investigated. Recently, a role has been proposed for neutralising antibody in conferring prevention/protection against HCMV infection/disease in pregnant women with primary HCMV infection. However, the level of cooperation between humoral immunity and the well-established T-cell protection remains to be defined.

  4. 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...

  5. D-4F, an apolipoprotein A-I mimetic, inhibits TGF-β1 induced epithelial-mesenchymal transition in human alveolar epithelial cell.

    PubMed

    You, Jia; Wang, Jintao; Xie, Linshen; Zhu, Chengwen; Xiong, Jingyuan

    2016-10-01

    Emerging evidences support that transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT) participates in the pathogenesis of pulmonary fibrosis and asthmatic airway remodeling. Recent studies demonstrated that apolipoprotein A-I (Apo A-I) is the only known substance that can resolve established pulmonary fibrotic nodules, and Apo A-I mimetic D-4F (a synthetic polypeptide consisting of 18 amino acids) plays an inhibitory role in murine asthmatic model. However, cellular mechanisms for such therapeutic effects of Apo A-I and D-4F remain to be elucidated. This study evaluated the effects of D-4F on TGF-β1 induced EMT in human type II alveolar epithelial cell line A549. A549 cells treated with 10ng/ml of TGF-β1 manifested distinct EMT, including fibroblastic morphological changes, down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. These EMT related changes were all inhibited by D-4F in a concentration dependent manner. Transcriptional investigation demonstrated clearly that D-4F dose-dependently compensated for the reduced E-cadherin mRNA level and the increased vimentin mRNA level in TGF-β1 treated A549 cells. Translational analysis revealed that D-4F significantly reversed the TGF-β1 induced changes of E-cadherin and vimentin levels. These results suggested that D-4F inhibits TGF-β1 induced EMT in human alveolar epithelial cell. Given the functional similarities between D-4F and Apo A-I, it is speculated that D-4F and Apo A-I are able to exert possible anti-fibrotic and anti-asthmatic effects via inhibiting alveolar EMT, and D-4F may possess beneficial clinical potential for patients suffering from pulmonary fibrosis and asthma.

  6. VAV1 represses E-cadherin expression through the transactivation of Snail and Slug: a potential mechanism for aberrant epithelial to mesenchymal transition in human epithelial ovarian cancer.

    PubMed

    Wakahashi, Senn; Sudo, Tamotsu; Oka, Noriko; Ueno, Sayaka; Yamaguchi, Satoshi; Fujiwara, Kiyoshi; Ohbayashi, Chiho; Nishimura, Ryuichiro

    2013-09-01

    Ovarian cancer is the most lethal gynecological malignancy in the western world. Although patients with early-stage ovarian cancer generally have a good prognosis, approximately 20%-30% of patients will die of the disease, and 5-year recurrence rates are 25%-45%, highlighting the need for improved detection and treatment. We investigated the role of VAV1, a protein with guanine nucleotide exchange factor activity, which is associated with survival in patients with early-stage ovarian cancer (International of Obstetrics and Gynecology [FIGO] stages I and II). We analyzed 88 samples from patients with primary epithelial ovarian cancer, which were divided into FIGO stages I and II (n = 46), and III and IV (n = 42). Prognostic analysis revealed that upregulated VAV1 expression correlated significantly with poor prognosis in patients with early-stage epithelial ovarian cancer (P ≤ 0.05), but not with other clinicopathologic features. Stable overexpression of VAV1 in human high-grade serous ovarian cancer SKOV3 cells induced morphologic changes indicative of loss of intercellular adhesions and organized actin stress fibers. Western blotting and real-time reverse transcriptase-polymerase chain reaction demonstrated that these cells had downregulated E-cadherin protein and messenger RNA levels, respectively. This downregulation is associated with epithelial-mesenchymal transition (EMT) and invasive cancer. Furthermore, VAV1 overexpression in both SKOV3 and human ovarian surface epithelial cells demonstrated that its upregulation of an E-cadherin transcriptional repressor, Snail and Slug, was not confined to ovarian cancer cells. Conversely, knockdown of VAV1 by RNA interference reduced Snail and Slug. Our findings suggest that VAV1 may play a role in the EMT of ovarian cancer, and may serve as a potential therapeutic target.

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

    SciTech Connect

    Feltens, Ralph; Moegel, Iljana; Roeder-Stolinski, Carmen; Simon, Jan-Christoph; Herberth, Gunda; Lehmann, Irina

    2010-01-01

    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 emphasis 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.

  8. Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

    PubMed Central

    Sumitomo, Tomoko; Nakata, Masanobu; Higashino, Miharu; Yamaguchi, Masaya; Kawabata, Shigetada

    2016-01-01

    Group A Streptococcus (GAS) is a human-specific pathogen responsible for local suppurative and life-threatening invasive systemic diseases. Interaction of GAS with human plasminogen (PLG) is a salient characteristic for promoting their systemic dissemination. In the present study, a serotype M28 strain was found predominantly localized in tricellular tight junctions of epithelial cells cultured in the presence of PLG. Several lines of evidence indicated that interaction of PLG with tricellulin, a major component of tricellular tight junctions, is crucial for bacterial localization. A site-directed mutagenesis approach revealed that lysine residues at positions 217 and 252 within the extracellular loop of tricellulin play important roles in PLG-binding activity. Additionally, we demonstrated that PLG functions as a molecular bridge between tricellulin and streptococcal surface enolase (SEN). The wild type strain efficiently translocated across the epithelial monolayer, accompanied by cleavage of transmembrane junctional proteins. In contrast, amino acid substitutions in the PLG-binding motif of SEN markedly compromised those activities. Notably, the interaction of PLG with SEN was dependent on PLG species specificity, which influenced the efficiency of bacterial penetration. Our findings provide insight into the mechanism by which GAS exploits host PLG for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions. PMID:26822058

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

    SciTech Connect

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

    1997-07-01

    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 proteins 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.

  10. Transport of stearic acid-based solid lipid nanoparticles (SLNs) into human epithelial cells.

    PubMed

    Shah, Rohan M; Rajasekaran, Dhivya; Ludford-Menting, Mandy; Eldridge, Daniel S; Palombo, Enzo A; Harding, Ian H

    2016-04-01

    Development of drug delivery systems, as much as the drug molecule itself, is an important consideration for improving drug absorption and bioavailability. The mechanisms by which drug carriers enter target cells can differ depending on their size, surface properties and components. Solid lipid nanoparticles (SLNs) have gained an increased attention in recent years and are the drug carriers of interest in this paper. They are known to breach the cell-membrane barrier and have been actively sought to transport biomolecules. Previous studies by our group, and also other groups, provided an extensive characterization of SLNs. However, few studies have investigated the uptake of SLNs and these have had limited mechanistic focus. The aim of this work was to investigate the pathway of uptake of SLNs by human epithelial cells i.e., lung A549 and cervical HeLa cells. To the best of our knowledge, this is first study that investigates the cellular uptake of SLNs by human epithelial cells. The mechanism of cellular uptake was deciphered using pharmacologic inhibitors (sucrose, potassium-free buffer, filipin and cytochalasin B). Imaging techniques and flow assisted cell sorting (FACS) were used to assess the cellular uptake of SLNs loaded with rhodamine 123 as a fluorescent probe. This study provided evidence that the cellular uptake of SLNs was energy-dependent, and the endocytosis of SLNs was mainly dependent on clathrin-mediated mechanisms. The establishment of entry mechanism of SLNs is of fundamental importance for future facilitation of SLNs as biological or drug carriers.

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

    PubMed

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

    1989-10-01

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

  12. Toxicity of Topical Antifungal Agents to Stratified Human Cultivated Corneal Epithelial Sheets

    PubMed Central

    Kimakura, Mikiko; Yokoo, Seiichi; Nakagawa, Suguru; Yamagami, Satoru; Amano, Shiro

    2014-01-01

    Abstract Purpose: Prolonged use of topical antifungal agents may compromise corneal epithelial integrity. Here, we used an in vitro model of human stratified corneal epithelium to compare the ocular toxicity profiles of 4 different antifungal eye drops. Methods: Human corneal epithelial cell sheets were cultured in a serum-free medium containing 0.1% micafungin, 1% voriconazole, 5% pimaricin, 0.1% amphotericin B, or controls (saline or 5% glucose). Cell viability and barrier function were measured by WST-1 assay and carboxyfluorescein permeability assay, respectively. Cell migration was measured on a wound healing assay. Results: WST-1 assay and carboxyfluorescein permeability assay revealed that amphotericin B was the most toxic drug, followed by pimaricin, micafungin, and voriconazole. Cell migration on a wound healing assay was decreased in the following order, amphotericin B, pimaricin, micafungin, and voriconazole. Conclusions: Topical micafungin and voriconazole appeared to be the least toxic to the corneal epithelium. Drug prescription should consider not only fungal species and susceptibility but also ocular toxicity and stage of treatment. PMID:25280055

  13. 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

  14. Lycium barbarum polysaccharides protected human retinal pigment epithelial cells against oxidative stress-induced apoptosis

    PubMed Central

    Liu, Lian; Lao, Wei; Ji, Qing-Shan; Yang, Zhi-Hao; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2015-01-01

    AIM To investigate the protective effect and its mechanism of lycium barbarum polysaccharides (LBP) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells. METHODS ARPE-19 cells, a human retinal pigment epithelial cell lines, were exposed to different concentrations of H2O2 for 24h, then cell viability was measured by Cell Counting Kit-8 (CCK-8) assay to get the properly concentration of H2O2 which can induce half apoptosis of APRE-19. With different concentrations of LBP pretreatment, the ARPE-19 cells were then exposed to appropriate concentration of H2O2, cell apoptosis was detected by flow cytometric analysis. Expression levels of Bcl-2 and Bax were measured by real time quantitative polymerase chain reaction (RT-PCR) technique. RSULTS LBP significantly reduced the H2O2-induced ARPE-19 cells' apoptosis. LBP inhibited the H2O2-induced down-regulation of Bcl-2 and up-regulation of Bax. CONCLUSION LBP could protect ARPE-19 cells from H2O2-induced apoptosis. The Bcl-2 family had relationship with the protective effects of LBP. PMID:25709900

  15. Cigarette smoke alters primary human bronchial epithelial cell differentiation at the air-liquid interface.

    PubMed

    Schamberger, Andrea C; Staab-Weijnitz, Claudia A; Mise-Racek, Nikica; Eickelberg, Oliver

    2015-02-02

    The differentiated human airway epithelium consists of different cell types forming a polarized and pseudostratified epithelium. This is dramatically altered in chronic obstructive pulmonary disease (COPD), characterized by basal and goblet cell hyperplasia, and squamous cell metaplasia. The effect of cigarette smoke on human bronchial epithelial cell (HBEC) differentiation remains to be elucidated. We analysed whether cigarette smoke extract (CSE) affected primary (p)HBEC differentiation and function. pHBEC were differentiated at the air-liquid interface (ALI) and differentiation was quantified after 7, 14, 21, or 28 days by assessing acetylated tubulin, CC10, or MUC5AC for ciliated, Clara, or goblet cells, respectively. Exposure of differentiating pHBEC to CSE impaired epithelial barrier formation, as assessed by resistance measurements (TEER). Importantly, CSE exposure significantly reduced the number of ciliated cells, while it increased the number of Clara and goblet cells. CSE-dependent cell number changes were reflected by a reduction of acetylated tubulin levels, an increased expression of the basal cell marker KRT14, and increased secretion of CC10, but not by changes in transcript levels of CC10, MUC5AC, or FOXJ1. Our data demonstrate that cigarette smoke specifically alters the cellular composition of the airway epithelium by affecting basal cell differentiation in a post-transcriptional manner.

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

    PubMed

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

    2004-12-01

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

  17. Carbon nanotubes induce apoptosis resistance of human lung epithelial cells through FLICE-inhibitory protein.

    PubMed

    Pongrakhananon, Varisa; Luanpitpong, Sudjit; Stueckle, Todd A; Wang, Liying; Nimmannit, Ubonthip; Rojanasakul, Yon

    2015-02-01

    Chronic exposure to single-walled carbon nanotubes (SWCNT) has been reported to induce apoptosis resistance of human lung epithelial cells. As resistance to apoptosis is a foundation of neoplastic transformation and cancer development, we evaluated the apoptosis resistance characteristic of the exposed lung cells to understand the pathogenesis mechanism. Passage control and SWCNT-transformed human lung epithelial cells were treated with known inducers of apoptosis via the intrinsic (antimycin A and CDDP) or extrinsic (FasL and TNF-α) pathway and analyzed for apoptosis by DNA fragmentation, annexin-V expression, and caspase activation assays. Whole-genome microarray was performed to aid the analysis of apoptotic gene signaling network. The SWCNT-transformed cells exhibited defective death receptor pathway in association with cellular FLICE-inhibitory protein (c-FLIP) overexpression. Knockdown or chemical inhibition of c-FLIP abrogated the apoptosis resistance of SWCNT-transformed cells. Whole-genome expression signature analysis confirmed these findings. This study is the first to demonstrate carbon nanotube-induced defective death receptor pathway and the role of c-FLIP in the process.

  18. Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.

    PubMed

    Cho, Myung Soo; Kim, Sang Jin; Ku, Seung-Yup; Park, Jung Hyun; Lee, Haksup; Yoo, Dae Hoon; Park, Un Chul; Song, Seul Ae; Choi, Young Min; Yu, Hyeong Gon

    2012-09-01

    Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.

  19. 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.

  20. Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray.

    PubMed

    Abiko, Yoshimitsu; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Tsushima, Katsumasa; Ohta, Mitsuhiro; Sasahara, Hiroshige

    2004-03-01

    Gingival epithelial cells and fibroblasts play important roles and have a harmonious relationship under normal and disease conditions, but the precise differences between theses cells remain unknown. To study the differences in gene expression between human gingival epithelial cells (HGE) and human gingival fibroblasts (HGF), mRNA was recovered from primary cultured cells and analyzed using cDNA microarray technology. The cDNA retro-transcribed from equal quantities of mRNA was labeled with the fluorescent dyes Cy5 and Cy3. The mixed probes were then hybridized with 7276 genes on the DNA microarray, after which fluorescence signals were scanned and further analyzed using GeneSpring software. Of the 7276 genes screened, 469 showed expression levels that were more than 2-fold greater in HGE than in HGF, while 293 showed expression levels that were more than 2-fold greater in HGF than in HGE. To confirm the reliability of the microarray results, keratin K5 and desmocolin, and vimentin and gp130, which showed higher mRNA levels in HGE and HGF, respectively, were selected and their mRNA levels were further analyzed by RT-PCR. The results of RT-PCR correlated well with those of microarray analysis. The present findings using a DNA microarray to detect differences in the gene expression profiles of HGE and HGF may be beneficial for genetic diagnosis of periodontal tissue metabolism and periodontal diseases.

  1. 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.

  2. Bifidobacteria Prevent Tunicamycin-Induced Endoplasmic Reticulum Stress and Subsequent Barrier Disruption in Human Intestinal Epithelial Caco-2 Monolayers

    PubMed Central

    Akiyama, Takuya; Oishi, Kenji

    2016-01-01

    Endoplasmic reticulum (ER) stress is caused by accumulation of unfolded and misfolded proteins in the ER, thereby compromising its vital cellular functions in protein production and secretion. Genome wide association studies in humans as well as experimental animal models linked ER stress in intestinal epithelial cells (IECs) with intestinal disorders including inflammatory bowel diseases. However, the mechanisms linking the outcomes of ER stress in IECs to intestinal disease have not been clarified. In this study, we investigated the impact of ER stress on intestinal epithelial barrier function using human colon carcinoma-derived Caco-2 monolayers. Tunicamycin-induced ER stress decreased the trans-epithelial electrical resistance of Caco-2 monolayers, concomitant with loss of cellular plasma membrane integrity. Epithelial barrier disruption in Caco-2 cells after ER stress was not caused by caspase- or RIPK1-dependent cell death but was accompanied by lysosomal rupture and up-regulation of the ER stress markers Grp78, sXBP1 and Chop. Interestingly, several bifidobacteria species inhibited tunicamycin-induced ER stress and thereby diminished barrier disruption in Caco-2 monolayers. Together, these results showed that ER stress compromises the epithelial barrier function of Caco-2 monolayers and demonstrate beneficial impacts of bifidobacteria on ER stress in IECs. Our results identify epithelial barrier loss as a potential link between ER stress and intestinal disease development, and suggest that bifidobacteria could exert beneficial effects on this phenomenon. PMID:27611782

  3. Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

    PubMed Central

    Epa, Amali P.; Thatcher, Thomas H.; Pollock, Stephen J.; Wahl, Lindsay A.; Lyda, Elizabeth; Kottmann, R. M.; Phipps, Richard P.; Sime, Patricia J.

    2015-01-01

    Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation. Measurements and Main Results In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E2 in lung epithelial cells, and a PGE2 neutralizing antibody blocked the protective effect of epithelial cell co-culture. Conclusions We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE2. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis

  4. Human thymic epithelial primary cells produce exosomes carrying tissue-restricted antigens

    PubMed Central

    Skogberg, Gabriel; Lundberg, Vanja; Berglund, Martin; Gudmundsdottir, Judith; Telemo, Esbjörn; Lindgren, Susanne; Ekwall, Olov

    2015-01-01

    Exosomes are nano-sized vesicles released by cells into the extracellular space and have been shown to be present in thymic tissue both in mice and in humans. The source of thymic exosomes is however still an enigma and hence it is not known whether thymic epithelial cells (TECs) are able to produce exosomes. In this work, we have cultured human TECs and isolated exosomes. These exosomes carry tissue-restricted antigens (TRAs), for example, myelin basic protein and desmoglein 3. The presence of TRAs indicates a possible role for thymic epithelium-derived exosomes in the selection process of thymocytes. The key contribution of these exosomes could be to disseminate self-antigens from the thymic epithelia, thus making them more accessible to the pool of maturing thymocytes. This would increase the coverage of TRAs within the thymus, and facilitate the process of positive and negative selection. PMID:25776846

  5. 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

  6. 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.

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

    SciTech Connect

    Moonen, Harald J.J. . E-mail: h.moonen@grat.unimaas.nl; Geraets, Liesbeth; Vaarhorst, Anika; Bast, Aalt; Wouters, Emiel F.M.; Hageman, Geja J.

    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 enzyme 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.

  8. Paraquat induces epithelial-mesenchymal transition-like cellular response resulting in fibrogenesis and the prevention of apoptosis in human pulmonary epithelial cells.

    PubMed

    Yamada, Atsushi; Aki, Toshihiko; Unuma, Kana; Funakoshi, Takeshi; Uemura, Koichi

    2015-01-01

    The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ) poisoning. Epithelial-mesenchymal transition (EMT) has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β) is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0-500 μM) for 2-12 days. Short-term (2 days) high-dose (>100 μM) treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker), suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion). In contrast, long-term (6-12 days) low-dose (30 μM) treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA). The mesenchymal-like cells also secreted the extracellular matrix (ECM) protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE) cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during subacute PQ

  9. Antiapoptotic effects of estrogen in normal and cancer human cervical epithelial cells.

    PubMed

    Wang, Qifang; Li, Xin; Wang, Liqin; Feng, Ying-Hong; Zeng, Robin; Gorodeski, George

    2004-12-01

    The present study investigated the antiapoptotic effects of estrogen in normal and cancer human cervical cells and the mechanisms involved. Baseline apoptosis in human cervical epithelial cells is mediated predominantly by P2X7-receptor-induced, Ca(2+)-dependent activation of the mitochondrial (caspase-9) pathway. Treatment with 10 nM 17beta-estradiol blocked apoptosis induced by the P2X7-receptor ligands ATP and 2',3'-0-(4-benzoylbenzoyl)-ATP in normal human cervical epithelial cells (hECEs) and attenuated the effect in hECEs immortalized with human papillomavirus-16 (ECE16-1) and the cancer cervical cells HT3 and CaSki. Diethylstilbestrol and to a lesser degree estrone could mimic the effects of 17beta-estradiol, whereas actinomycin-D and cycloheximide attenuated the response. The antiapoptotic effect of estrogen did not depend on cell cycle phase, and in both normal and cancer cervical cells, it involved attenuation of activation of caspase-9 and the terminal caspase-3. However, involvement of cascades upstream to the caspase-9 differed in normal vs. cancer cervical cells. In the normal hECEs estrogen blocked P2X7-receptor-induced calcium influx. In contrast, in the cancer CaSki cells, estrogen up-regulated expression of Bcl-2 and attenuated Ca(2+)-induced mitochondrial swelling (i.e. formation of mitochondrial permeability transition pores). Estrogen had no effect on P2X7-receptor-induced apoptosis in the anaplastic SiHa and Hela cells. These results point to a novel antiapoptotic effect of estrogen in the cervix that is independent of its mitogenic function. The results also suggest that cancer cervical cells evolved antiapoptotic mechanisms that enable the cells to evade apoptosis and could therefore promote tumor progression.

  10. Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells.

    PubMed

    Khanna, Sugandha; Darbre, Philippa D

    2013-05-01

    Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben resulted in a greater number of colonies per dish (P < 0.05 in each case) and an increased average colony size (P < 0.001 in each case). Dose-responses showed that concentrations as low as 10(-6) M methylparaben, 10(-7) M n-propylparaben and 10(-7) M n-butylparaben could increase colony numbers (P = 0.016, P = 0.010, P = 0.008, respectively): comparison with a recent measurement of paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis.

  11. Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells.

    PubMed

    Cerri, Chiara; Chimenti, Daniele; Conti, Ilaria; Neri, Tommaso; Paggiaro, Pierluigi; Celi, Alessandro

    2006-08-01

    Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

  12. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology.

    PubMed

    Shaharuddin, Bakiah; Ahmad, Sajjad; Md Latar, Nani; Ali, Simi; Meeson, Annette

    2016-10-14

    : Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue.

  13. Comparison of cadmium cytotoxicity in human versus rat nasal epithelial cells in vitro

    SciTech Connect

    Morgan, D.L.; Humphreys, J.E.; Bilotta, J.M.; Nixon, J.C.; Hatch, G.E.

    1990-01-01

    Quantitative extrapolation of toxicity data from animals to humans will allow more accurate assessments of human health risks. The overall objective of this investigation is to provide the tissue sensitivity data necessary to extrapolate quantitatively the toxic effects of inhaled particles from animals to humans. In the initial study, cadmium sulfate (CdSO4) toxicity for human and Fischer 344 rat nasal turbinate epithelial (NTE) cells was evaluated in vitro. The studies were unique in that both rat and human NTE cells were obtained from fresh, normal tissue. Methods were developed for isolating and culturing NTE cells from rat and human tissue using identical procedures, and for measuring the cellular nucleotides by HPLC. Changes in adenylate energy charge (EC) and nucleotide levels were used as toxicity endpoints. Cellular Cd levels were measured by graphite furnace atomic absorption spectrometry and expressed as microgram Cd/microgram DNA. Using these methods, the cellular Cd dose and the toxic effect in each cell type were measured and compared.

  14. IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells

    PubMed Central

    Adams, Kelly M.; Abraham, Valsamma; Spielman, Daniel; Kolls, Jay K.; Rubenstein, Ronald C.; Conner, Gregory E.; Cohen, Noam A.; Kreindler, James L.

    2014-01-01

    The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease. PMID:25141009

  15. Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

    PubMed Central

    Gowers, Kate H. C.; Lee, Dani Do Hyang; Brown, James M.; Crowley, Claire; Teixeira, Vitor H.; Smith, Claire M.; Urbani, Luca; Hamilton, Nicholas J.; Thakrar, Ricky M.; Booth, Helen L.; Birchall, Martin A.; De Coppi, Paolo; Giangreco, Adam; O’Callaghan, Christopher

    2016-01-01

    Rationale: Stem cell–based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell–seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air–liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air–liquid interface cultures. Measurements and Main Results: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Conclusions: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical

  16. N-acetylcysteine inhibits Na+ absorption across human nasal epithelial cells.

    PubMed

    Rochat, Thierry; Lacroix, Jean-Silvain; Jornot, Lan

    2004-10-01

    N-acetylcysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders. The mechanism of action is based on rupture of the disulfide bridges of the high molecular glycoproteins present in the mucus, resulting in smaller subunits of the glycoproteins and reduced viscosity of the mucus. Because Na(+) absorption regulates airway surface liquid volume and thus the efficiency of mucociliary clearance, we asked whether NAC affects the bioelectric properties of human nasal epithelial cells. A 24-h basolateral treatment with 10 mM of NAC decreased the transepithelial potential difference and short-circuit current (I(SC)) by 40%, and reduced the amiloride-sensitive current by 50%, without affecting the transepithelial resistance. After permeabilization of the basolateral membranes of cells with amphotericin B in the presence of a mucosal-to-serosal Na(+) gradient (135:25 mM), NAC inhibited 45% of the amiloride-sensitive current. The Na(+)-K(+)-ATPase pump activity and the basolateral K(+) conductance were not affected by NAC treatment. NAC did not alter total cell mRNA and protein levels of alpha-epithelial Na(+) channel (EnaC) subunit, but reduced abundance of alpha-ENaC subunits in the apical cell membrane as quantified by biotinylation. This effect can be ascribed to the sulphydryl (SH) group of NAC, since N-acetylserine and S-carboxymethyl-l-cysteine were ineffective. Given the importance of epithelial Na(+) channels in controlling the thin layer of fluid that covers the surface of the airways, the increase in the fluidity of the airway mucus following NAC treatment in vivo might be in part related to downregulation of Na(+) absorption and consequently water transport.

  17. Trichomonas vaginalis induces cytopathic effect on human lung alveolar basal carcinoma epithelial cell line A549.

    PubMed

    Salvador-Membreve, Daile Meek C; Jacinto, Sonia D; Rivera, Windell L

    2014-12-01

    Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genitourinary tract. However, several case reports with supporting molecular and immunological identifications have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports have documented that its occurrence is associated with respiratory failures. The medical significance or consequence of this association is unclear. Thus, to establish the possible outcome from the interaction of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cultures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron microscopy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549 epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50-72.4%) to A549 and this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of incubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 monolayer cultures. Further studies are recommended to completely elucidate this association.

  18. UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells

    PubMed Central

    Black, Adrienne T.; Gordon, Marion. K.; Heck, Diane E.; Gallo, Michael A.; Laskin, Debra L.; Laskin, Jeffrey D.

    2011-01-01

    The cornea is highly sensitive to ultraviolet B (UVB) light-induced oxidative stress, a process that results in the production of inflammatory mediators which have been implicated in tissue injury. In the present studies, we characterized the inflammatory response of human corneal epithelial cells to UVB (2.5 - 25 mJ/cm2). UVB caused a dose-dependent increase in the generation of reactive oxygen species in the cells. This was associated with increases in mRNA expression of the antioxidants Cu,Zn superoxide dismutase (SOD), Mn-SOD, catalase and heme oxygenase-1 (HO-1), as well as the glutathione S-transferases (GST), GSTA1-2, GSTA3, GSTA4, GSTM1, mGST2. UVB also upregulated expression of the proinflammatory cytokines, IFNγ, IL-1β, TGFβ and TNFα, and enzymes important in prostaglandin (PG) biosynthesis including cyclooxygenase-2 (COX-2) and the PG synthases mPGES-2, PGDS, PGFS and thromboxane synthase, and in leukotriene biosynthesis including 5-lipoxygenase (5-LOX), and the epidermal and platelet forms of 12-LOX and 15-LOX-2. UVB was found to activate JNK and p38 MAP kinases in corneal epithelial cells; ERK1/2 MAP kinase is constitutively active, but its activity was increased following UVB treatment. Inhibition of p38 blocked UVB-induced expression of TNFα, COX-2, PGDS and 15-LOX-2, while JNK inhibition suppressed TNFα and HO-1. These data indicate that UVB modulates corneal epithelial cell expression of antioxidants and proinflammatory mediators by distinct mechanisms. Alterations in expression of these mediators are likely to be important in regulating inflammation and protecting the cornea from UVB-induced oxidative stress. PMID:21300015

  19. Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells.

    PubMed

    Zsembery, Akos; Boyce, Amanda T; Liang, Lihua; Peti-Peterdi, János; Bell, P Darwin; Schwiebert, Erik M

    2003-04-11

    Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.

  20. Free fucose is a danger signal to human intestinal epithelial cells.

    PubMed

    Chow, Wai Ling; Lee, Yuan Kun

    2008-03-01

    Fucose is present in foods, and it is a major component of human mucin glycoproteins and glycolipids. l-Fucose can also be found at the terminal position of many cell-surface oligosaccharide ligands that mediate cell-recognition and adhesion-signalling pathways. Mucin fucose can be released through the hydrolytic activity of pathogens and indigenous bacteria, leading to the release of free fucose into the intestinal lumen. The immunomodulating effects of free fucose on intestinal epithelial cells (enterocyte-like Caco-2) were investigated. It was found that the presence of l-fucose up regulated genes and secretion of their encoded proteins that are involved in both the innate and adaptive immune responses, possibly via the toll-like receptor-2 signalling pathway. These include TNFSF5, TNFSF7, TNF-alpha, IL12, IL17 and IL18. Besides modulating immune reactions in differentiated Caco-2 cells, fucose induced a set of cytokine genes that are involved in the development and proliferation of immune cells. These include the bone morphogenetic proteins (BMP) BMP2, BMP4, IL5, thrombopoietin and erythropoietin. In addition, the up regulated gene expression of fibroblast growth factor-2 may help to promote epithelial cell restitution in conjunction with the enhanced expression of transforming growth factor-beta mRNA. Since the exogenous fucose was not metabolised by the differentiated Caco-2 cells as a carbon source, the reactions elicited were suggested to be a result of the direct interaction of fucose and differentiated Caco-2 cells. The presence of free fucose may signal the invasion of mucin-hydrolysing microbial cells and breakage of the mucosal barrier. The intestinal epithelial cells respond by up regulation and secretion of cytokines, pre-empting the actual invasion of pathogens.

  1. Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells.

    PubMed

    Nabokina, Svetlana M; Ramos, Mel Brendan; Valle, Judith E; Said, Hamid M

    2015-05-01

    Microbiota of the large intestine synthesize considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). There is a specific high-affinity regulated carrier-mediated uptake system for TPP in human colonocytes (product of the SLC44A4 gene). The mechanisms of regulation of SLC44A4 gene expression are currently unknown. In this study, we characterized the SLC44A4 minimal promoter region and identified transcription factors important for basal promoter activity in colonic epithelial cells. The 5'-regulatory region of the SLC44A4 gene (1,022 bp) was cloned and showed promoter activity upon transient transfection into human colonic epithelial NCM460 cells. With the use of a series of 5'- and 3'-deletion luciferase reporter constructs, the minimal genomic region that required basal transcription of the SLC44A4 gene expression was mapped between nucleotides -178 and +88 (using the distal transcriptional start site as +1). Mutational analysis performed on putative cis-regulatory elements established the involvement of ETS/ELF3 [E26 transformation-specific sequence (ETS) proteins], cAMP-responsive element (CRE), and SP1/GC-box sequence motifs in basal SLC44A4 promoter activity. By means of EMSA, binding of ELF3 and CRE-binding protein-1 (CREB-1) transcription factors to the SLC44A4 minimal promoter was shown. Contribution of CREB into SLC44A4 promoter activity was confirmed using NCM460 cells overexpressing CREB. We also found high expression of ELF3 and CREB-1 in colonic (NCM460) compared with noncolonic (ARPE19) cells, suggesting their possible contribution to colon-specific pattern of SLC44A4 expression. This study represents the first characterization of the SLC44A4 promoter and reports the importance of both ELF3 and CREB-1 transcription factors in the maintenance of basal promoter activity in colonic epithelial cells.

  2. Calpain 1 regulates TGF-β1-induced epithelial-mesenchymal transition in human lung epithelial cells via PI3K/Akt signaling pathway

    PubMed Central

    Tan, Wei-Jun; Tan, Qiu-Yue; Wang, Ting; Lian, Min; Zhang, Li; Cheng, Zhen-Shun

    2017-01-01

    Cell proliferation, transformation, and epithelial-mesenchymal transition (EMT) are key processes involved in the development of idiopathic pulmonary fibrosis (IPF). This study investigated the regulatory factors and signaling pathways that mediate EMT in the human type II alveolar epithelial A549 cell line. A549 cells were cultured in RPMI-1640 medium and allocated to the following four groups: blank control group or treated with transforming growth factor-β1 (TGF-β1), TGF-β1 + PD 150606 (a calpain 1 inhibitor), or PD 150606. We examined E-cadherin (E-cad), α-smooth muscle actin (α-SMA), and calpain 1 mRNA transcript and protein expression levels in these four groups by performing RT-PCR and western blot analyses. The results indicated that TGF-β1 treatment significantly downregulated E-cad and upregulated α-SMA expression compared with that of the blank control group (P<0.05). TGF-β1 also enhanced calpain 1 expression compared with that of the blank control group (P<0.05). By contrast, treatment with the calpain 1 inhibitor PD 150606 increased E-cad expression and decreased α-SMA expression. Furthermore, PD 150606 treatment antagonized TGF-β1-mediated increase in Akt/phospho-Akt in A549 epithelial cells. However, TGF-β1-induced ETM was not correlated with the ERK and JNK signaling pathways. These combined results indicate that calpain 1 could regulate EMT in TGF-β1-treated A549 epithelial cells via the PI3K/Akt signaling pathway. PMID:28386365

  3. Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

    PubMed Central

    Centeno, A; Davis, C P; Cohen, M S; Warren, M M

    1983-01-01

    The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells. Images PMID:6132878

  4. Stromal-epithelial paracrine interactions in the neoplastic rat and human prostate.

    PubMed

    Djakiew, D; Pflug, B; Onoda, M

    1993-01-01

    Homotypic paracrine interactions in the rat and human prostate have been investigated using prostatic stromal cells and neoplastic epithelial cells (PA-III, rat; TSU-pr1, human). Secretory proteins prepared from each cell type were used to determine the dose dependent regulation of growth (DNA synthesis) of the corresponding homotypic responder cell, as determined by 3H-thymidine incorporation. PA-III secretory protein stimulated rat stromal cell proliferation by 1.8-fold. This stimulatory activity of PA-III protein on stromal cell proliferation was partially reduced (approximately 35%) by treatment with nerve growth factor (NGF) antibody, whereas neither acidic fibroblast growth factor (aFGF) antibody nor basic fibroblast growth factor (bFGF) antibody immunoneutralized the stimulatory activity of PA-III cell protein. In the corresponding opposite interaction, rat stromal cell protein modulated PA-III growth in a biphasic manner. At lower concentrations of stromal cell protein (1.25 micrograms/ml) PA-III cell growth was stimulated by 1.6-fold, whereas at higher concentrations of protein (100 micrograms/ml) PA-III cell growth was inhibited to 60%. Treatment of the stromal cell protein (1.25 micrograms/ml and 100 micrograms/ml) with NGF antibody reduced PA-III cell relative growth to approximately 30% and 5%, respectively. bFGF antibody treatment of stromal cell protein at 1.25 micrograms/ml did not influence relative growth, whereas bFGF antibody treatment of 100 micrograms/ml stromal cell protein reduced relative growth by an additional 40%. Treatment of the stromal cell protein (1.25 micrograms/ml and 100 micrograms/ml) with aFGF antibodies reduced relative growth from that observed at these two protein concentrations by approximately 50% in both cases. Human epithelial TSU-pr1 protein stimulated human stromal cell proliferation approximately 1.7-fold. Treatment of TSU-pr1 protein with NGF antibody resulted in stimulation of human stromal cell proliferation (4

  5. Using Organotypic Epithelial Tissue Culture to Study the Human Papillomavirus Life Cycle.

    PubMed

    Lee, Denis; Norby, Kathryn; Hayes, Mitchell; Chiu, Ya-Fang; Sugden, Bill; Lambert, Paul F

    2016-05-06

    Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent "dead ends" for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus. In order to study the full viral life cycle, from the establishment to maintenance to productive stages, our lab makes use of the organotypic epithelial tissue culture system. This system allows us to mimic the three-dimensional structure of epithelia whose differentiation is tightly linked to the completion of the HPV viral life cycle. In this chapter we describe how various aspects of the HPV life cycle are monitored in raft cultures making use of an immortalized keratinocyte cell line. © 2016 by John Wiley & Sons, Inc.

  6. Procyanidin A2 Modulates IL-4-Induced CCL26 Production in Human Alveolar Epithelial Cells

    PubMed Central

    Coleman, Sara L.; Kruger, Marlena C.; Sawyer, Gregory M.; Hurst, Roger D.

    2016-01-01

    Allergic asthma is an inflammatory lung disease that is partly sustained by the chemokine eotaxin-3 (CCL26), which extends eosinophil migration into tissues long after allergen exposure. Modulation of CCL26 could represent a means to mitigate airway inflammation. Here we evaluated procyanidin A2 as a means of modulating CCL26 production and investigated interactions with the known inflammation modulator, Interferon γ (IFNγ). We used the human lung epithelial cell line A549 and optimized the conditions for inducing CCL26. Cells were exposed to a range of procyanidin A2 or IFNγ concentrations for varied lengths of time prior to an inflammatory insult of interleukin-4 (IL-4) for 24 h. An enzyme-linked immunosorbent assay was used to measure CCL26 production. Exposing cells to 5 μM procyanidin A2 (prior to IL-4) reduced CCL26 production by 35% compared with control. Greatest inhibition by procyanidin A2 was seen with a 2 h exposure prior to IL-4, whereas IFNγ inhibition was greatest at 24 h. Concomitant incubation of procyanidin A2 and IFNγ did not extend the inhibitory efficacy of procyanidin A2. These data provide evidence that procyanidin A2 can modulate IL-4-induced CCL26 production by A549 lung epithelial cells and that it does so in a manner that is different from IFNγ. PMID:27845745

  7. Acid and organic aerosol coatings on magnetic nanoparticles increase iron concentrations in human airway epithelial cells.

    PubMed

    Ghio, Andrew J; Dailey, Lisa A; Richards, Judy H; Jang, Myoseon

    2009-07-01

    Numerous industrial applications for man-made nanoparticles have been proposed. Interactions of nanoparticles with agents in the atmosphere may impact human health. We tested the postulate that in vitro exposures of respiratory epithelial cells to airborne magnetic nanoparticles (MNP; Fe(3)O(4)) with and without a secondary organic aerosol (SOA) and an inorganic acid could affect iron homeostasis, oxidative stress, and interleukin (IL)-8 release. Cell iron concentrations were increased after exposures to MNP and values were further elevated with co-exposures to either SOA or inorganic acid. Increased expression of ferritin and elevated levels of RNA for DMT1, proteins for iron storage and transport respectively, followed MNP exposures, but values were significant for only those with co-exposures to inorganic acid and organic aerosols. Cell iron concentration corresponded to a measure of oxidative stress in the airway epithelial cells; MNP with co-exposures to SOA and inorganic acid increased both available metal and indices of oxidant generation. Finally, the release of a proinflammatory cytokine (i.e. IL-8) by the exposed cells similarly increased with cell iron concentration. We conclude that MNP can interact with a SOA and an inorganic acid to present metal in a catalytically reactive state to cultured respiratory cells. This produces an oxidative stress to affect a release of IL-8.

  8. 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

  9. The Diacetyl-exposed Human Airway Epithelial Secretome: New Insights Into Flavoring Induced Airways Disease.

    PubMed

    Brass, David M; Gwinn, William M; Valente, Ashlee M; Kelly, Francine L; Brinkley, Christie D; Nagler, Andrew E; Moseley, M Arthur; Morgan, Daniel L; Palmer, Scott M; Foster, Matthew W

    2017-03-01

    Bronchiolitis obliterans (BO) is an increasingly important lung disease characterized by fibroproliferative airway lesions and decrements in lung function. Occupational exposure to the artificial food flavoring ingredient diacetyl, commonly used to impart a buttery flavor to microwave popcorn, has been associated with BO development. In the occupational setting, diacetyl vapor is first encountered by the airway epithelium. To better understand the effects of diacetyl vapor on the airway epithelium we used an unbiased proteomic approach to characterize both the apical and basolateral secretomes of air liquid interface cultures of primary human airway epithelial cells from four unique donors after exposure to an occupationally relevant ~1100 ppm of diacetyl vapor or PBS as a control on alternating days. Basolateral and apical supernatants collected 48 hours after the third exposure were analyzed using one-dimensional liquid chromatography tandem mass spectrometry. Paired t-tests adjusted for multiple comparisons were used to assess differential expression between diacetyl and PBS exposure. Of the significantly differentially expressed proteins identified, 61 were unique to the apical secretome, 81 were unique to the basolateral secretome and there were an additional 11 present in both. Pathway enrichment analysis using publicly available databases reveals that proteins associated with matrix remodeling including degradation, assembly and new matrix organization were over-represented in the data sets. Similarly, protein modifiers of epidermal growth factor receptor signaling were significantly altered. The ordered changes in protein expression suggest that the airway epithelial response to diacetyl may contribute to BO pathogenesis.

  10. Osteogenic Differentiation of Human Amniotic Epithelial Cells and Its Application in Alveolar Defect Restoration

    PubMed Central

    Jiawen, Si; Jianjun, Zhang; Jiewen, Dai; Dedong, Yu; Hongbo, Yu; Jun, Shi; Xudong, Wang; Shen, Steve G.F.

    2014-01-01

    The present study investigated the detailed in vitro osteogenic differentiation process and in vivo bone regenerative property of human amniotic epithelial cells (hAECs). The in vitro osteogenic differentiation process of hAECs was evaluated by biochemical staining, real-time polymerase chain reaction, and immunofluorescence. Next, β-tricalcium phosphate (β-TCP) scaffolds alone or loaded with hAECs were implanted into the alveolar defects of rats. Micro-computed tomography evaluation and histologic studies were conducted. Our results validated the in vitro osteogenic capacity of hAECs by upregulation of Runx2, osterix, alkaline phosphatase, collagen I, and osteopontin, with positive biochemical staining for osteoblasts. An epithelial-mesenchymal transformation process might be involved in the osteogenic differentiation of hAECs by increased expression of transforming growth factor-β1. Our data also demonstrated that in vivo implantation of hAECs loaded on β-TCP scaffolds, not only improved bone regeneration by direct participation, but also reduced the early host immune response to the scaffolds. The presented data indicate that hAECs possess proper osteogenic differentiation potential and a modulatory influence on the early tissue remodeling process, making these cells a potential source of progenitor cells for clinical restoration of the alveolar defect. PMID:25368378

  11. MEF up-regulates human beta-defensin 2 expression in epithelial cells.

    PubMed

    Lu, Zhuo; Kim, Kyoung-Ah; Suico, Mary Ann; Shuto, Tsuyoshi; Li, Jian-Dong; Kai, Hirofumi

    2004-03-12

    Human beta-defensin 2 (HBD2), an antimicrobial peptide, is widely expressed in epithelial tissues and displays a potent killing activity in response to the invasiveness of a wide range of microorganisms and the stimulation of various molecules. Myeloid ELF-1-like factor (MEF) has been reported to be involved in innate immunity responses, such as activation of perforin and lysozyme transcription. The role of MEF in the transcription regulation of HBD2, however, is unknown. Here, we show that MEF not only activated HBD2 promoter activity, but also increased the endogenous HBD2 transcription level. Moreover, the activated HBD2 promoter activity was attenuated by the antisense MEF RNA input and the loss of the ETS binding site (EBS: GGAA core sequence) in the HBD2 promoter. The interaction between the EBS and MEF protein was further confirmed by electrophoretic mobility shift assay. Thus, our data indicate that MEF may play an important role in regulating HBD2 expression in epithelial cells.

  12. Barrier responses of human bronchial epithelial cells to grass pollen exposure.

    PubMed

    Blume, Cornelia; Swindle, Emily J; Dennison, Patrick; Jayasekera, Nivenka P; Dudley, Sarah; Monk, Phillip; Behrendt, Heidrun; Schmidt-Weber, Carsten B; Holgate, Stephen T; Howarth, Peter H; Traidl-Hoffmann, Claudia; Davies, Donna E

    2013-07-01

    The airway epithelium forms a physical, chemical and immunological barrier against inhaled environmental substances. In asthma, these barrier properties are thought to be abnormal. In this study, we analysed the effect of grass pollen on the physical and immunological barrier properties of differentiated human primary bronchial epithelial cells. Following exposure to Timothy grass (Phleum pratense) pollen extract, the integrity of the physical barrier was not impaired as monitored by measuring the transepithelial resistance and immunofluorescence staining of tight junction proteins. In contrast, pollen exposure affected the immunological barrier properties by modulating vectorial mediator release. CXC chemokine ligand (CXCL)8/interleukin (IL)-8 showed the greatest increase in response to pollen exposure with preferential release to the apical compartment. Inhibition of the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways selectively blocked apical CXCL8/IL-8 release via a post-transcriptional mechanism. Apical release of CC chemokine ligand (CCL)20/macrophage inflammatory protein-3α, CCL22/monocyte-derived chemokine and tumour necrosis factor-α was significantly increased only in severe asthma cultures, while CCL11/eotaxin-1 and CXCL10/interferon-γ-induced protein-10 were reduced in nonasthmatic cultures. The bronchial epithelial barrier modulates polarised release of mediators in response to pollen without direct effects on its physical barrier properties. The differential response of cells from normal and asthmatic donors suggests the potential for the bronchial epithelium to promote immune dysfunction in asthma.

  13. Focal adhesion kinase knockdown modulates the response of human corneal epithelial cells to topographic cues.

    PubMed

    Dreier, Britta; Raghunathan, Vijaya Krishna; Russell, Paul; Murphy, Christopher J

    2012-12-01

    A rapidly expanding literature broadly documents the impact of biophysical cues on cellular behaviors. In spite of increasing research efforts in this field, the underlying signaling processes are poorly understood. One of the candidate molecules for being involved in mechanotransduction is focal adhesion kinase (FAK). To examine the role of FAK in the response of immortalized human corneal epithelial (hTCEpi) cells to topographic cues, FAK was depleted by siRNA transfection. Contrary to expectations, FAK knockdown resulted in an enhanced response with a greater number of hTCEpi cells aligned to the long axis of anisotropically ordered surface ridges and grooves. Both underlying topographic features and FAK depletion modulated the migration of corneal epithelial cells. The impact of FAK knockdown on both migration and alignment varied depending on the topographic cues to which the cells were exposed, with the most significant change observed on the biologically relevant size scale (400nm). Additionally, a change in expression of genes encoding perinuclear Nesprins 1 and 2 (SYNE1, 2) was observed in response to topographic cues. SYNE1/2 expression was also altered by FAK depletion, suggesting that these proteins might represent a link between cytosolic and nuclear signaling processes. The data presented here have relevance to our understanding of the fundamental processes involved in corneal cell behavior to topographic cues. These results highlight the importance of incorporating biophysical cues in the conduction of in vitro studies and into the design and fabrication of implantable prosthetics.

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

    SciTech Connect

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena; Martinez-Irujo, Juan Jose; Rouzaut, Ana

    2008-05-01

    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 emitted 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.

  15. 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.

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

    PubMed

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

    2015-11-16

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

  17. Serum-Induced Keratinization Processes in an Immortalized Human Meibomian Gland Epithelial Cell Line

    PubMed Central

    Hampel, Ulrike; Schröder, Antje; Mitchell, Todd; Brown, Simon; Snikeris, Peta; Garreis, Fabian; Kunnen, Carolina; Willcox, Mark; Paulsen, Friedrich

    2015-01-01

    Purpose The aim of this study was to evaluate a human meibomian gland epithelial cell line (HMGEC) as a model for meibomian gland (patho)physiology in vitro. Methods HMGEC were cultured in the absence or presence of serum. Sudan III lipid staining, ultrastructural analysis and lipidomic analyses were performed. Impedance sensing, desmoplakin 1/2 mRNA and cytokeratin (CK) 1, 5, 6, 14 levels were evaluated. Serum containing medium supplemented with higher serum, glucose, an omega-3 lipid cocktail, eicosapentaenoic acid or sebomed medium were investigated for lipid accumulation and ultrastructural morphology. Results Lipid droplet accumulation in HMGEC was induced by serum containing media after 1 day, but decreased over time. Cultivation in serum induced desmosome and cytokeratin filament formation. Desmoplakin 1/2 gene levels were significantly upregulated after 1d of serum treatment. Furthermore, the normalized impedance increased significantly. Lipidome analysis revealed high levels of phospholipids (over 50%), but very low levels of wax ester and cholesteryl esters (under 1%). Stimulation with eicosapentaenoic acid increased lipid accumulation after one day. Conclusion Serum treatment of HMGEC caused lipid droplet formation to some extent but also induced keratinization. The cells did not produce typical meibum lipids under these growth conditions. HMGEC are well suited to study (hyper)keratinization processes of meibomian gland epithelial cells in vitro. PMID:26042605

  18. 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

  19. Intestinal epithelial culture under an air-liquid interface: a tool for studying human and mouse esophagi.

    PubMed

    Yokobori, T; Suzuki, S; Miyazaki, T; Sohda, M; Sakai, M; Tanaka, N; Ozawa, D; Hara, K; Honjo, H; Altan, B; Fukuchi, M; Ishii, H; Iwatsuki, M; Sugimachi, K; Sudo, T; Iwaya, T; Nishida, N; Mimori, K; Kuwano, H; Mori, M

    2016-10-01

    This study investigated whether an intestinal epithelial culture method can be applied to mouse and human esophageal cultures. The esophagi harvested from 1-day-old mice and adult humans were maintained in collagen gels. A commercially available culture medium for human embryonic stem cells was used for the human esophageal culture. We discovered that the intestinal epithelial culture method can be successfully applied to both mouse and human esophageal cultures. The long-term cultured esophageal organoids were rod-like luminal structures lined with myofibroblasts. We discovered that regeneration of the esophageal mucosal surface can be almost completely achieved in vitro, and the advantage of this method is that organoid cultures may be generated using host-derived fibroblasts as a niche. This method is a promising tool for mouse and human research in intestinal biology, carcinogenesis, and regenerative medicine.

  20. Evidence for a stromal-epithelial “lactate shuttle” in human tumors

    PubMed Central

    Lin, Zhao; Ertel, Adam; Flomenberg, Neal; Witkiewicz, Agnieszka K; Birbe, Ruth C; Howell, Anthony; Pavlides, Stephanos; Gandara, Ricardo; Pestell, Richard G; Sotgia, Federica

    2011-01-01

    Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal

  1. The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

    PubMed Central

    Malliri, Angeliki; Symons, Marc; Hennigan, Robert F.; Hurlstone, Adam F.L.; Lamb, Richard F.; Wheeler, Tricia; Ozanne, Bradford W.

    1998-01-01

    Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion. PMID:9817764

  2. Melatonin as a negative mitogenic hormonal regulator of human prostate epithelial cell growth: potential mechanisms and clinical significance.

    PubMed

    Tam, Chun W; Chan, Kwok W; Liu, Vincent W S; Pang, Bo; Yao, Kwok-Ming; Shiu, Stephen Y W

    2008-11-01

    Circannual variation in the human serum levels of prostate-specific antigen, a growth marker of the prostate gland, has been reported recently. The present study was conducted to investigate the role of the photoperiodic hormone melatonin (MLT) and its membrane receptors in the modulation of human prostate growth. Expression of MT(1) and MT(2) receptors was detected in benign human prostatic epithelial tissues and RWPE-1 cells. MLT and 2-iodomelatonin inhibited RWPE-1 cell proliferation and up-regulated p27(Kip1) gene and protein expression in the cells. The effects of MLT were blocked by the nonselective MT(1)/MT(2) receptor antagonist luzindole, but were not affected by the selective MT(2) receptor antagonist 4-phenyl-2-propionamidotetraline. Of note, the antiproliferative action of MLT on benign prostate epithelial RWPE-1 cells was effected via increased p27(Kip1) gene transcription through MT(1) receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel, a signaling process which has previously been demonstrated in 22Rv1 prostate cancer cells. Taken together, the demonstration of the MT(1)/PKA+PKC/p27(Kip1) antiproliferative pathway in benign and malignant prostate epithelial cell lines indicated the potential importance of this MLT receptor-mediated signaling mechanism in growth regulation of the human prostate gland in health and disease. Collectively, our data support the hypothesis that MLT may function as a negative mitogenic hormonal regulator of human prostate epithelial cell growth.

  3. 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.

  4. Reciprocal Paracrine Interactions Between Normal Human Epithelial and Mesenchymal Cells Protect Cellular DNA from Radiation-Induced Damage

    SciTech Connect

    Nakazawa, Yuka; Saenko, Vladimir Rogounovitch, Tatiana; Suzuki, Keiji; Mitsutake, Norisato; Matsuse, Michiko; Yamashita, Shunichi

    2008-06-01

    Purpose: To explore whether interactions between normal epithelial and mesenchymal cells can modulate the extent of radiation-induced DNA damage in one or both types of cells. Methods and Materials: Human primary thyrocytes (PT), diploid fibroblasts BJ, MRC-5, and WI-38, normal human mammary epithelial cells (HMEC), and endothelial human umbilical cord vein endothelial cells (HUV-EC-C), cultured either individually or in co-cultures or after conditioned medium transfer, were irradiated with 0.25 to 5 Gy of {gamma}-rays and assayed for the extent of DNA damage. Results: The number of {gamma}-H2AX foci in co-cultures of PT and BJ fibroblasts was approximately 25% lower than in individual cultures at 1 Gy in both types of cells. Reciprocal conditioned medium transfer to individual cultures before irradiation resulted in approximately a 35% reduction of the number {gamma}-H2AX foci at 1 Gy in both types of cells, demonstrating the role of paracrine soluble factors. The DNA-protected state of cells was achieved within 15 min after conditioned medium transfer; it was reproducible and reciprocal in several lines of epithelial cells and fibroblasts, fibroblasts, and endothelial cells but not in epithelial and endothelial cells. Unlike normal cells, human epithelial cancer cells failed to establish DNA-protected states in fibroblasts and vice versa. Conclusions: The results imply the existence of a network of reciprocal interactions between normal epithelial and some types of mesenchymal cells mediated by soluble factors that act in a paracrine manner to protect DNA from genotoxic stress.

  5. Chemical carcinogen-induced decreases in genomic 5-methyldeoxycytidine content of normal human bronchial epithelial cells

    SciTech Connect

    Wilson, V.L.; Smith, R.A.; Longoria, J.; Liotta, M.A.; Harper, C.M.; Harris, C.C.

    1987-05-01

    The genomic content of DNA 5-methyldeoxycytidine (m/sup 5/dC) was measured in dividing normal human bronchial epithelial cells treated with a broad range of chemical carcinogens. At noncytotoxic concentrations, all of the carcinogenic agents tested significantly reduced cellular DNA m/sup 5/dC content whereas the weakly carcinogenic and noncarcinogenic agents, benzo(e)pyrene and phenanthrene (respectively), did not. These reductions varied from 8% to 31% depending on the agent and the donor cells. The reduction is genomic m/sup 5/dC levels were concentration dependent for the carcinogenic polycyclic aromatic hydrocarbon benzo(a)pyrene. The authors speculate that carcinogen-induced perturbation of DNA m/sup 5/dC patterns may lead to heritable changes in gene expression and contribute to the molecular alterations involved in the initiation and the subsequent steps of the carcinogenesis process.

  6. 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.

  7. Analysis of the activation routes induced by different metal oxide nanoparticles on human lung epithelial cells

    PubMed Central

    Simón-Vázquez, Rosana; Lozano-Fernández, Tamara; Dávila-Grana, Angela; González-Fernández, África

    2016-01-01

    Nanoparticles (Nps) can induce toxicity in the lung by accidental or intentional exposure. The main objective of the study reported here was to characterize the effect that four metal oxide Nps (CeO2, TiO2, Al2O3 and ZnO) had at the cellular level on a human lung epithelial cell line. This goal was achieved by studying the capacity of the Nps to activate the main mitogen-activated protein kinases (MAPKs) and the nuclear factor NFκB. Only ZnO Nps were able to activate all of the MAPKs and the release of Zn2+ ions was the main cause of activation. ZnO and Al2O3 Nps activated the NFκB pathway and induced the release of inflammatory cytokines. CeO2 and TiO2 Nps were found to have safer profiles. The graphical abstract was obtained using Servier Medical Art. PMID:28031965

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

    PubMed

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

    2015-03-10

    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.

  9. 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).

  10. Intracellular trafficking pathway of BK virus in human renal proximal tubular epithelial cells

    PubMed Central

    Moriyama, Takahito; Sorokin, Andrey

    2009-01-01

    Intracellular trafficking of BK Virus (BKV) in human renal proximal tubular epithelial cells (HRPTEC) is critical for BKV nephritis. However, the major trafficking components utilized by BKV remain unknown. Co-incubation of HRPTEC with BKV and microtubule disrupting agents prevented BKV infection as detected by immunofluorescence and western blot analysis with antibodies which recognize BKV large T antigen. However, inhibition of a dynein, cellular motor protein, did not interfere with BKV infection in HRPTEC. A colocalization study of BKV with the markers of the endoplasmic reticulum (ER) and the Golgi apparatus (GA), indicated that BKV reached the ER from 6 to 10 hours, while bypassing the GA or passing through the GA too transiently to be detected. This study contributes to the understanding of mechanisms of intracellular trafficking used by BKV in the infection of HRPTEC. PMID:17976677

  11. Continuous mucociliary transport by primary human airway epithelial cells in vitro

    PubMed Central

    Sears, Patrick R.; Yin, Wei-Ning

    2015-01-01

    Mucociliary clearance (MCC) is an important innate defense mechanism that continuously removes inhaled pathogens and particulates from the airways. Normal MCC is essential for maintaining a healthy respiratory system, and impaired MCC is a feature of many airway diseases, including both genetic (cystic fibrosis, primary ciliary dyskinesia) and acquired (chronic obstructive pulmonary disease, bronchiectasis) disorders. Research into the fundamental processes controlling MCC, therefore, has direct clinical application, but has been limited in part due to the difficulty of studying this complex multicomponent system in vitro. In this study, we have characterized a novel method that allows human airway epithelial cells to differentiate into a mucociliary epithelium that transports mucus in a continuous circular track. The mucociliary transport device allows the measurement and manipulation of all features of mucociliary transport in a controlled in vitro system. In this initial study, the effect of ciliary beat frequency and mucus concentration on the speed of mucociliary transport was investigated. PMID:25979076

  12. Upregulated CTHRC1 promotes human epithelial ovarian cancer invasion through activating EGFR signaling.

    PubMed

    Ye, Jun; Chen, Wei; Wu, Zhi-Yong; Zhang, Jin-Hui; Fei, He; Zhang, Li-Wen; Wang, Ya-Hui; Chen, Ya-Ping; Yang, Xiao-Mei

    2016-12-01

    Epithelial ovarian cancer (EOC) is the major cause of deaths from gynecologic malignancies, and metastasis is the main cause of cancer related death. Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein that has the ability to inhibit collagen matrix synthesis. In this study, we found that high CTHRC1 expression was associated with poor prognosis of EOC. In vitro experiments showed that CTHRC1 promoted migration and invasion of ovarian cancer cells. CTHRC1 had no effect on ovarian cancer cells viability. Additionally, EGFR inhibitors reduced the promotion effects of CTHRC1 on EOC cell invasion. After silencing of CTHRC1, downregulated expression of phosphorylation of EGFR/ERK1/2/AKT was observed in ovarian cancer cells. Taken together, our results suggest a role for CTHRC1 in the progression of ovarian cancer and identified CTHRC1 as a potentially important predictor for human ovarian cancer prognosis.

  13. 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.

  14. 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.

  15. 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.

  16. Molecular mechanisms of microRNAs in regulating epithelial-mesenchymal transitions in human cancers.

    PubMed

    Tang, Jinlong; Li, Yuan; Wang, Jingyu; Wen, Zhineng; Lai, Maode; Zhang, Honghe

    2016-02-28

    The epithelial-mesenchymal transition (EMT) provides a strong driving force in the progression of various human cancers and the development of chemoresistance. Recently, numbers of studies have demonstrated that microRNAs (miRNAs), by post-transcriptionally silencing EMT-related molecules, can promote or inhibit the EMT process and play pivotal roles in effectively manipulating the occurrence, development, invasion, and metastasis of cancers. MiRNAs can also control the EMT or be controlled by genetic modification and mutual regulation, especially negative feedback. Therefore, miRNAs can be viewed as either oncogenes or tumor suppressor genes to facilitate or retard the EMT, resulting in far-reaching impact on tumor metastasis and effective diagnosis, treatment, and prognosis.

  17. Characteristics of functionalized nano-hydroxyapatite and internalization by human epithelial cell

    NASA Astrophysics Data System (ADS)

    Yan-Zhong, Zhao; Yan-Yan, Huang; Jun, Zhu; Shai-Hong, Zhu; Zhi-You, Li; Ke-Chao, Zhou

    2011-11-01

    Hydroxyapatite is the main inorganic component of biological bone and tooth enamel, and synthetic hydroxyapatite has been widely used as biomaterials. In this study, a facile method has been developed for the fabrication of arginine-functionalized and europium-doped hydroxyapatite nanoparticles (Arg-Eu-HAP). The synthesized nanoparticles characterized by transmission electron microscopy, X-ray diffractometry, Fourier transform infrared, and Zeta potential analyzer. Its biological properties with DNA binding, cell toxicity, cell binding and intracellular distribution were tested by agarose gel electrophoresis assay, flow cytometry, and fluorescence microscope and laser scanning confocal microscope. The synthesized Arg-Eu-HAP could effectively bind DNA without any cytotoxicity and be internalized into the cytoplasm and perinuclear of human lung epithelial cells.

  18. NOTCH1 and NOTCH2 regulate epithelial cell proliferation in mouse and human gastric corpus.

    PubMed

    Demitrack, Elise S; Gifford, Gail B; Keeley, Theresa M; Horita, Nobukatsu; Todisco, Andrea; Turgeon, D Kim; Siebel, Christian W; Samuelson, Linda C

    2017-02-01

    The Notch signaling pathway is known to regulate stem cells and epithelial cell homeostasis in gastrointestinal tissues; however, Notch function in the corpus region of the stomach is poorly understood. In this study we examined the consequences of Notch inhibition and activation on cellular proliferation and differentiation and defined the specific Notch receptors functioning in the mouse and human corpus. Notch pathway activity was observed in the mouse corpus epithelium, and gene expression analysis revealed NOTCH1 and NOTCH2 to be the predominant Notch receptors in both mouse and human. Global Notch inhibition for 5 days reduced progenitor cell proliferation in the mouse corpus, as well as in organoids derived from mouse and human corpus tissue. Proliferation effects were mediated through both NOTCH1 and NOTCH2 receptors, as demonstrated by targeting each receptor alone or in combination with Notch receptor inhibitory antibodies. Analysis of differentiation by marker expression showed no change to the major cell lineages; however, there was a modest increase in the number of transitional cells coexpressing markers of mucous neck and chief cells. In contrast to reduced proliferation after pathway inhibition, Notch activation in the adult stomach resulted in increased proliferation coupled with reduced differentiation. These findings suggest that NOTCH1 and NOTCH2 signaling promotes progenitor cell proliferation in the mouse and human gastric corpus, which is consistent with previously defined roles for Notch in promoting stem and progenitor cell proliferation in the intestine and antral stomach.

  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. In Vitro Modeling of RSV Infection and Cytopathogenesis in Well-Differentiated Human Primary Airway Epithelial Cells (WD-PAECs).

    PubMed

    Broadbent, Lindsay; Villenave, Remi; Guo-Parke, Hong; Douglas, Isobel; Shields, Michael D; Power, Ultan F

    2016-01-01

    The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis.

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

    SciTech Connect

    Hirano, Seishiro; Fujitani, Yuji; Furuyama, Akiko; Kanno, Sanae

    2010-11-15

    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. Over 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.

  2. Curcumin inhibits proliferation of human lens epithelial cells: a proteomic analysis*

    PubMed Central

    Hu, Yan-hong; Huang, Xiu-rong; Qi, Ming-xin; Hou, Bu-yuan

    2012-01-01

    Objective: The incidence of after-cataracts [also known as posterior capsular opacification (PCO)] is between 30% and 50% three years following cataract surgery. Suppressing the proliferation of lens epithelial cells (LECs) is a primary goal in preventing PCO. Here, we investigated the proteomic regulation of the inhibitory effects of curcumin (Cur) on the proliferation of human lens epithelial B3 (HLE-B3) cells. Methods: Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 20 mg/L Cur in a CO2 incubator for 24 h. Results: We found that the absorbance (A) value of rhbFGF group was significantly higher than the A value of the control group. Furthermore, the A value of the Cur group was significantly lower compared to the rhbFGF group, with an inhibition of 53.7%. Five different protein spots were obtained from proliferative HLE-B3 cells induced by rhbFGF. Eight different protein spots were obtained in HLE-B3 cells incubated with Cur. There were the common variational protein spots at mass/charge (m/z) ratios of 8 093 and 13 767 between rhbFGF group and control group as well as between the Cur group and rhbFGF group. Conclusions: These results show that Cur effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. The protein spots at m/z of 8 093 and 13 767 may be the targets of Cur-induced inhibition of HLE-B3 cell proliferation. Cur may be a reliable and effective drug for prevention and treatment of polymerase chain reaction (PCR). PMID:22556179

  3. Human epithelial cells exposed to functionalized multiwalled carbon nanotubes: interactions and cell surface modifications.

    PubMed

    Fanizza, C; Casciardi, S; Incoronato, F; Cavallo, D; Ursini, C L; Ciervo, A; Maiello, R; Fresegna, A M; Marcelloni, A M; Lega, D; Alvino, A; Baiguera, S

    2015-09-01

    With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 μg/mL for 1, 2, 4 h; 5, 10, 20 μg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 μg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to

  4. Phototoxicity and cytotoxicity of fullerol in human retinal pigment epithelial cells

    SciTech Connect

    Wielgus, Albert R.; Zhao, Baozhong; Chignell, Colin F.; Hu, Dan-Ning; Roberts, Joan E.

    2010-01-01

    The water-soluble nanoparticle hydroxylated fullerene [fullerol, nano-C{sub 60}(OH){sub 22-26}] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have previously found that fullerol is both cytotoxic and phototoxic to human lens epithelial cells (HLE B-3) and that the endogenous antioxidant lutein blocked some of this phototoxicity. In the present study we have found that fullerol induces cytotoxic and phototoxic damage to human retinal pigment epithelial cells. Accumulation of nano-C{sub 60}(OH){sub 22-26} in the cells was confirmed spectrophotometrically at 405 nm, and cell viability, cell metabolism and membrane permeability were estimated using trypan blue, MTS and LDH assays, respectively. Fullerol was cytotoxic toward hRPE cells maintained in the dark at concentrations higher than 10 muM. Exposure to an 8.5 J.cm{sup -2} dose of visible light in the presence of > 5 muM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 muM lutein offered some protection against fullerol photodamage. Using time resolved photophysical techniques, we have now confirmed that fullerol produces singlet oxygen with a quantum yield of PHI = 0.05 in D{sub 2}O and with a range of 0.002-0.139 in various solvents. As our previous studies have shown that fullerol also produces superoxide in the presence of light, retinal phototoxic damage may occur through both type I (free radical) and type II (singlet oxygen) mechanisms. In conclusion, ocular exposure to fullerol, particularly in the presence of sunlight, may lead to retinal damage.

  5. Substance P induces CCN1 expression via histone deacetylase activity in human colonic epithelial cells.

    PubMed

    Koon, Hon Wai; Shih, David Q; Hing, Tressia C; Chen, Jeremy; Ho, Samantha; Zhao, Dezheng; Targan, Stephan R; Pothoulakis, Charalabos

    2011-11-01

    We have shown that substance P (SP) and its neurokinin-1 receptor (NK-1R) regulate intestinal angiogenesis by increasing expression of protein CYR61 (the cysteine-rich angiogenic inducer 61, or CCN1) in colonic epithelial cells. However, the mechanism involved in SP-induced CCN1 expression has not been studied, and the outcome of increased CCN1 expression in the development of colitis is not fully understood. Because histone deacetylase (HDAC) modulates transcription of several genes involved in inflammation, we investigated participation of HDAC in SP-induced CCN1 expression in human colonic epithelial NCM460 cells overexpressing NK-1R (NCM460-NK-1R) and in primary colonocytes. SP increased HDAC activity with deacetylation and dephosphorylation of nucleosome protein histone H3 in NCM460-NK-1R and/or primary colonocytes. Histone deacetylation and dephosphorylation was observed in colonic mucosa from irritable bowel disease patients. Similarly, colonic mucosal tissues from mice exposed to dextran sulfate sodium showed histone H3 deacetylation and dephosphorylation and increased HDAC activity that was reversed by the NK-1R antagonist CJ-12255. SP-induced increased CCN1 expression in NCM460-NK-1R cells was abolished by pharmacological HDAC inhibition. HDAC overexpression activated basal and SP-induced CCN1 promoter activity. Intracolonic CCN1 overexpression significantly ameliorated dextran sulfate sodium-induced colitis, with reduction of proinflammatory cytokine expression in mice. Thus, SP-mediated CCN1 expression in the inflamed human and mouse colon involves increased HDAC activity. Our results strongly suggest that increased CCN1 expression may be involved in mucosal healing during colitis.

  6. Human amnion epithelial cells modulate the inflammatory response to ventilation in preterm lambs

    PubMed Central

    Melville, Jacqueline M.; McDonald, Courtney A.; Bischof, Robert J.; Polglase, Graeme R.; Lim, Rebecca; Wallace, Euan M.; Jenkin, Graham; Moss, Timothy J.

    2017-01-01

    Ventilation of preterm neonates causes pulmonary inflammation that can contribute to lung injury, propagate systemically and result in long-term disease. Modulation of this initial response may reduce lung injury and its sequelae. We aimed to determine the effect of human amnion epithelial cells (hAECs) on immune activation and lung injury in preterm neonatal lambs. Preterm lambs received intratracheal hAECs (90x106) or vehicle, prior to 2 h of mechanical ventilation. Within 5 min of ventilation onset, lambs also received intravenous hAECs (90x106) or vehicle. Lung histology, bronchoalveolar lavage (BAL) cell phenotypes, and cytokine profiles were examined after 2 h of ventilation, and in unventilated controls. Histological indices of lung injury were higher than control, in vehicle-treated ventilated lambs but not in hAEC-treated ventilated lambs. Ventilation-induced pulmonary leukocyte recruitment was greater in hAEC-treated lambs than in vehicle-treated lambs. Lung IL-1β and IL-6 mRNA expression was higher in vehicle- and hAEC-treated ventilated lambs than in controls but IL-8 mRNA levels were greater than control only in vehicle-treated ventilated lambs. Numbers of CD44+ and CD21+ lymphocytes and macrophages from the lungs were altered in vehicle- and hAEC-treated ventilated lambs. Numbers of CD8+ macrophages were lower in hAEC-treated ventilated lambs than in vehicle-treated ventilated lambs. Indices of systemic inflammation were not different between vehicle- and hAEC-treated lambs. Human amnion epithelial cells modulate the pulmonary inflammatory response to ventilation in preterm lambs, and reduce acute lung injury. Immunomodulatory effects of hAECs reduce lung injury in preterm neonates and may protect against longer-term respiratory disease. PMID:28346529

  7. 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

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

    PubMed Central

    Aldhahrani, Adil; Verdon, Bernard; 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. PMID:28344983

  10. Human amnion epithelial cells modulate the inflammatory response to ventilation in preterm lambs.

    PubMed

    Melville, Jacqueline M; McDonald, Courtney A; Bischof, Robert J; Polglase, Graeme R; Lim, Rebecca; Wallace, Euan M; Jenkin, Graham; Moss, Timothy J

    2017-01-01

    Ventilation of preterm neonates causes pulmonary inflammation that can contribute to lung injury, propagate systemically and result in long-term disease. Modulation of this initial response may reduce lung injury and its sequelae. We aimed to determine the effect of human amnion epithelial cells (hAECs) on immune activation and lung injury in preterm neonatal lambs. Preterm lambs received intratracheal hAECs (90x106) or vehicle, prior to 2 h of mechanical ventilation. Within 5 min of ventilation onset, lambs also received intravenous hAECs (90x106) or vehicle. Lung histology, bronchoalveolar lavage (BAL) cell phenotypes, and cytokine profiles were examined after 2 h of ventilation, and in unventilated controls. Histological indices of lung injury were higher than control, in vehicle-treated ventilated lambs but not in hAEC-treated ventilated lambs. Ventilation-induced pulmonary leukocyte recruitment was greater in hAEC-treated lambs than in vehicle-treated lambs. Lung IL-1β and IL-6 mRNA expression was higher in vehicle- and hAEC-treated ventilated lambs than in controls but IL-8 mRNA levels were greater than control only in vehicle-treated ventilated lambs. Numbers of CD44+ and CD21+ lymphocytes and macrophages from the lungs were altered in vehicle- and hAEC-treated ventilated lambs. Numbers of CD8+ macrophages were lower in hAEC-treated ventilated lambs than in vehicle-treated ventilated lambs. Indices of systemic inflammation were not different between vehicle- and hAEC-treated lambs. Human amnion epithelial cells modulate the pulmonary inflammatory response to ventilation in preterm lambs, and reduce acute lung injury. Immunomodulatory effects of hAECs reduce lung injury in preterm neonates and may protect against longer-term respiratory disease.

  11. 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.

  12. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells

    PubMed Central

    Couvigny, Benoît; de Wouters, Tomas; Kaci, Ghalia; Jacouton, Elsa; Delorme, Christine; Doré, Joël; Renault, Pierre; Blottière, Hervé M.

    2015-01-01

    The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB) in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor), we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health. PMID:25946041

  13. 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.

  14. Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis

    SciTech Connect

    Bao, X.; Sinha, M. |; Liu, T.; Hong, C.; Luxon, B.A. |; Garofalo, R.P. ||; Casola, A. ||

    2008-04-25

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

  15. Effect of Hyperosmolality on β-Defensin Gene Expression by Human Corneal Epithelial Cells

    PubMed Central

    Narayanan, Srihari; Manning, Jennifer; Proske, Rita; McDermott, Alison M.

    2008-01-01

    Purpose As human β-defensins (hBD) are important antimicrobial peptides at epithelial surfaces, including the ocular surface, we tested the effect of hyperosmolar conditions on the expression of these peptides by human corneal epithelial cells (HCECs). Methods Simian virus 40–transformed HCECs (n = 5) or primary cultured HCECs (n = 5) were treated with serum-free media or serum-free hyperosmolar (400–500 mOsm/kg) media for 24 hours or serum-free 500 mOsm/kg media for 12 to 48 hours. The effect of hyperosmolality on interleukin-1β (IL-1β)–induced hBD-2 expression was also tested. IL-6 expression was studied as a marker of IL-1β function. Expression of hBD-1, -2, and -3 and IL-6 mRNA was detected by reverse transcription–polymerase chain reaction (RT-PCR). The levels of active IL-1β (culture supernatants and cell lysates) and pro–IL-1β (cell lysates) were detected by enzyme-linked immunosorbent assay. Results HCECs constitutively expressed hBD-1 and -3 but not hBD-2. Hyperosmolar media had no effect on the basal expression of hBD-1 or -3 and did not induce the expression of hBD-2. Treatment with 500 mOsm/kg media for 24 hours decreased the ability of IL-1β to upregulate hBD-2 and IL-6 expression. Active or pro–IL-1β was not detected in any cell culture sample. Conclusion Our results suggest that the hyperosmolar environment observed in diseases such as dry eye does not alter defensin expression. However, a hyperosmolar environment may influence cytokine function in ocular surface cells and thus affect their response to injury and inflammation. PMID:17133055

  16. Sperm fucosyltransferase-5 mediates spermatozoa-oviductal epithelial cell interaction to protect human spermatozoa from oxidative damage.

    PubMed

    Huang, Venus Wenxin; Lee, Cheuk-Lun; Lee, Yin-Lau; Lam, Kevin K W; Ko, Jennifer K Y; Yeung, William S B; Ho, Pak-Chung; Chiu, Philip C N

    2015-06-01

    Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. Interaction between spermatozoa and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. Our previous data showed that oviductal epithelial cell membrane proteins interact with the human spermatozoa and protect them from ROS-induced reduction in sperm motility, membrane integrity and DNA integrity. Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human spermatozoa. In this study, we demonstrate for the first time that sFUT5 is involved in human spermatozoa-oviduct interaction and the beneficial effects of such interaction on the fertilizing ability of human spermatozoa. Anti-sFUT5 antibody-treated spermatozoa had reduced binding to oviductal membrane proteins. It is consistent with the result that affinity-purified sFUT5 is bound to the epithelial lining of human oviduct and to the immortalized human oviductal epithelial cell line, OE-E6/E7. Pretreatment of spermatozoa with anti-sFUT5 antibody and oviductal membrane proteins with sFUT5 suppressed the protective action of oviductal membrane proteins against ROS/cryopreservation-induced oxidative damage in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of oviductal epithelial cell membrane proteins on sperm motility, membrane and DNA integrity. The results enhance our understanding on the protective mechanism of oviduct on sperm functions.

  17. 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

  18. 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.

  19. 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,...

  20. CO-EXPOSURE OF HUMAN AIRWAY EPITHELIAL CELLS TO OZONE AND PARTICULATE MATTER: EFFECTS ON ARACHIDONIC ACID METABOLISM

    EPA Science Inventory

    Co-exposure of human airway epithelial cells to ozone and particulate matter: effects on arachidonic acid metabolism.

    D. Stamm1, L. Dailey2, M.C. Madden2
    1 University of North Carolina-Chapel Hill, School of Medicine
    2 U.S. EPA, ORD, NHEERL, HSD, Chapel Hill, NC, USA...

  1. Modulation of Kingella kingae Adherence to Human Epithelial Cells by Type IV Pili, Capsule, and a Novel Trimeric Autotransporter

    PubMed Central

    Porsch, Eric A.; Kehl-Fie, Thomas E.; Geme, Joseph W. St.

    2012-01-01

    ABSTRACT Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. Colonization of the posterior pharynx is a key step in the pathogenesis of K. kingae disease. Previous work established that type IV pili are necessary for K. kingae adherence to the respiratory epithelium. In this study, we set out to identify additional factors that influence K. kingae interactions with human epithelial cells. We found that genetic disruption of the gene encoding a predicted trimeric autotransporter protein called Knh (Kingella NhhA homolog) resulted in reduced adherence to human epithelial cells. In addition, we established that K. kingae elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called ctrABCD for surface presentation. Furthermore, we discovered that the presence of a surface capsule interferes with Knh-mediated adherence to human epithelial cells by nonpiliated organisms and that maximal adherence in the presence of a capsule requires the predicted type IV pilus retraction machinery, PilT/PilU. On the basis of the data presented here, we propose a novel adherence mechanism that allows K. kingae to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance. PMID:23093386

  2. Activation of eicosanoid metabolism in human airway epithelial cells by ozonolysis products of membrane fatty acids.

    PubMed

    Leikauf, G D; Zhao, Q; Zhou, S; Santrock, J

    1995-09-01

    Inhaled ozone can react with a variety of cellular macromolecules within the lung. Recent analyses of the chemistry of ozone reactions with unsaturated fatty acids, which are present in all membranes and in mucus in the airways, indicate that ozonolysis yields one aldehyde and one hydroxyhydroperoxide molecule for each molecule of ozone. The hydroxyhydroperoxide molecule is unstable in aqueous environments, and subsequently yields a second aldehyde and hydrogen peroxide. The structure of common unsaturated fatty acids is such that attack by ozone at the carbon-carbon double bonds will yield 3-, 6-, and 9-carbon saturated and unsaturated aldehydes and hydroxyhydroperoxide. This study examines the effects of ozonolysis products on eicosanoid metabolism in human airway epithelial cells. Eicosanoid biosynthesis is important in a wide array of pathophysiological responses in the airway, and the release of eicosanoids by the epithelial barrier is likely to be significant in diseases induced by environmental factors. Previously, we demonstrated that ozone can increase eicosanoid synthesis from airway epithelial cells exposed in vitro. Human exposures to concentrations of ozone below the current National Ambient Air Quality Standard (0.12 ppm, not to be exceeded for more than one hour once per year) also resulted in increased eicosanoids in bronchoalveolar lavage fluid. To determine whether ozonolysis products could activate eicosanoid release, we exposed human airway epithelial cells to 3-, 6-, and 9-carbon aldehydes, hydroxyhydroperoxides, and hydrogen peroxide. We measured (1) eicosanoid metabolism using high-performance liquid chromatography and radioimmunoassays, and (2) the effects of the aldehydes, hydroxyhydroperoxides, and hydrogen peroxide on cell lysis. Eicosanoid release increased after exposure to aldehyde; release induced by 9-carbon (nonanal) aldehyde was greater than that induced by the 6-carbon (hexanal) or 3-carbon (propanal) aldehydes

  3. 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.

  4. 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.

  5. Effects of light-emitting diode radiations on human retinal pigment epithelial cells in vitro.

    PubMed

    Chamorro, Eva; Bonnin-Arias, Cristina; Pérez-Carrasco, María Jesús; Muñoz de Luna, Javier; Vázquez, Daniel; Sánchez-Ramos, Celia

    2013-01-01

    Human visual system is exposed to high levels of natural and artificial lights of different spectra and intensities along lifetime. Light-emitting diodes (LEDs) are the basic lighting components in screens of PCs, phones and TV sets; hence it is so important to know the implications of LED radiations on the human visual system. The aim of this study was to investigate the effect of LEDs radiations on human retinal pigment epithelial cells (HRPEpiC). They were exposed to three light-darkness (12 h/12 h) cycles, using blue-468 nm, green-525 nm, red-616 nm and white light. Cellular viability of HRPEpiC was evaluated by labeling all nuclei with DAPI; Production of reactive oxygen species (ROS) was determined by H2DCFDA staining; mitochondrial membrane potential was quantified by TMRM staining; DNA damage was determined by H2AX histone activation, and apoptosis was evaluated by caspases-3,-7 activation. It is shown that LED radiations decrease 75-99% cellular viability, and increase 66-89% cellular apoptosis. They also increase ROS production and DNA damage. Fluorescence intensity of apoptosis was 3.7% in nonirradiated cells and 88.8%, 86.1%, 83.9% and 65.5% in cells exposed to white, blue, green or red light, respectively. This study indicates three light-darkness (12 h/12 h) cycles of exposure to LED lighting affect in vitro HRPEpiC.

  6. Atrazine affects phosphoprotein and protein expression in MCF-10A human breast epithelial cells.

    PubMed

    Huang, Peixin; Yang, John; Song, Qisheng

    2014-10-01

    Atrazine, a member of the 2-chloro-s-triazine family of herbicides, is the most widely used pesticide in the world and often detected in agriculture watersheds. Although it was generally considered as an endocrine disruptor, posing a potential threat to human health, the molecular mechanisms of atrazine effects remain unclear. Using two-dimensional gel electrophoresis, we identified a panel of differentially expressed phosphoproteins and total proteins in human breast epithelial MCF-10A cells after being exposed to environmentally relevant concentrations of atrazine. Atrazine treatments for 6 h resulted in differential expression of 4 phosphoproteins and 8 total-proteins as compared to the control cells (>1.5-fold, p<0.05). MALDI-TOF MS/MS analysis revealed that the differentially expressed proteins belong to various cellular compartments (nucleus, cytosol, membrane) and varied in function, including those regulating the stress response such as peroxiredoxin I, HSP70 and HSP27; structural proteins such as tropomyosin and profilin 1; and oncogenesis proteins such as ANP32A. Six of the 12 identified proteins were verified by quantitative PCR for their transcript levels. The most up-regulated phosphoprotein by atrazine treatment, ANP32A, was further analyzed for its expression, distribution and cellular localization using Western blot and immunocytochemical approaches. The results revealed that ANP32 expression after atrazine treatment increased dose and time dependently and was primarily located in the nucleus. This study may provide new evidence on the potential toxicity of atrazine in human cells.

  7. Genotoxic effects of five polycyclic aromatic hydrocarbons in human and rat mammary epithelial cells

    SciTech Connect

    Mane, S.S.; Purnell, D.M.; Hsu, Ih-chang )

    1990-01-01

    Five polycyclic aromatic hydrocarbons (PAHs) of different carcinogenic activities were evaluated for their effects on DNA synthesis ({sup 3}HTdR labeling index (L.I.)) of rat and human mammary epithelial cells (MEC) and for their effects on chromosomes in MEC-mediated sister chromatid exchange (SCE) assays. When compared with DMSO-treated cells, exposures of rat MEC to the two most potent carcinogens, i.e., 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(a)P), resulted in a 45-62% reduction in the L.I. of rat MEC. Another carcinogen, 20-methylcholanthrene (MCA), produced a 35-48% reduction in L.I., while the noncarcinogenic PAHs, 1,2-benzanthracene (BA) and benzo(e)pyrene (B(e)P), showed no effect. Similarly, exposures of human MEC to DMBA and B(a)P resulted in a 50-90% depression in L.I. while BA was significantly less effective. When co-cultivated with Chinese hamster V-79 cells in the presence of PAH, both rat and human MEC can activate and release the active metabolites to induce SCE in V-79 cells. Comparing depression of L.I., SCE, and in vivo carcinogenicity for the 5 PAHs, SCE mediated by rat MEC is better correlated with carcinogenicity in rat than L.I. depression.

  8. 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

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

    PubMed

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

    2003-05-15

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

  10. Dung biomass smoke activates inflammatory signaling pathways in human small airway epithelial cells.

    PubMed

    McCarthy, Claire E; Duffney, Parker F; Gelein, Robert; Thatcher, Thomas H; Elder, Alison; Phipps, Richard P; Sime, Patricia J

    2016-12-01

    Animal dung is a biomass fuel burned by vulnerable populations who cannot afford cleaner sources of energy, such as wood and gas, for cooking and heating their homes. Exposure to biomass smoke is the leading environmental risk for mortality, with over 4,000,000 deaths each year worldwide attributed to indoor air pollution from biomass smoke. Biomass smoke inhalation is epidemiologically associated with pulmonary diseases, including chronic obstructive pulmonary disease (COPD), lung cancer, and respiratory infections, especially in low and middle-income countries. Yet, few studies have examined the mechanisms of dung biomass smoke-induced inflammatory responses in human lung cells. Here, we tested the hypothesis that dung biomass smoke causes inflammatory responses in human lung cells through signaling pathways involved in acute and chronic lung inflammation. Primary human small airway epithelial cells (SAECs) were exposed to dung smoke at the air-liquid interface using a newly developed, automated, and reproducible dung biomass smoke generation system. The examination of inflammatory signaling showed that dung biomass smoke increased the production of several proinflammatory cytokines and enzymes in SAECs through activation of the activator protein (AP)-1 and arylhydrocarbon receptor (AhR) but not nuclear factor-κB (NF-κB) pathways. We propose that the inflammatory responses of lung cells exposed to dung biomass smoke contribute to the development of respiratory diseases.

  11. Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

    SciTech Connect

    Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.; Bell, Matthew W.; Waalkes, Michael P.; Tokar, Erik J.

    2015-07-01

    Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a

  12. 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.

  13. Clarifying CB2 receptor-dependent and independent effects of THC on human lung epithelial cells

    SciTech Connect

    Sarafian, Theodore Montes, Cindy; Harui, Airi; Beedanagari, Sudheer R.; Kiertscher, Sylvia; Stripecke, Renata; Hossepian, Derik; Kitchen, Christina; Kern, Rita; Belperio, John; Roth, Michael D.

    2008-09-15

    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 epithelial 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 ATP loss

  14. OVOL2 Maintains the Transcriptional Program of Human Corneal Epithelium by Suppressing Epithelial-to-Mesenchymal Transition.

    PubMed

    Kitazawa, Koji; Hikichi, Takafusa; Nakamura, Takahiro; Mitsunaga, Kanae; Tanaka, Azusa; Nakamura, Masahiro; Yamakawa, Tatsuya; Furukawa, Shiori; Takasaka, Mieko; Goshima, Naoki; Watanabe, Akira; Okita, Keisuke; Kawasaki, Satoshi; Ueno, Morio; Kinoshita, Shigeru; Masui, Shinji

    2016-05-10

    In development, embryonic ectoderm differentiates into neuroectoderm and surface ectoderm using poorly understood mechanisms. Here, we show that the transcription factor OVOL2 maintains the transcriptional program of human corneal epithelium cells (CECs), a derivative of the surface ectoderm, and that OVOL2 may regulate the differential transcriptional programs of the two lineages. A functional screen identified OVOL2 as a repressor of mesenchymal genes to maintain CECs. Transduction of OVOL2 with several other transcription factors induced the transcriptional program of CECs in fibroblasts. Moreover, neuroectoderm derivatives were found to express mesenchymal genes, and OVOL2 alone could induce the transcriptional program of CECs in neural progenitors by repressing these genes while activating epithelial genes. Our data suggest that the difference between the transcriptional programs of some neuroectoderm- and surface ectoderm-derivative cells may be regulated in part by a reciprocally repressive mechanism between epithelial and mesenchymal genes, as seen in epithelial-to-mesenchymal transition.

  15. Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features

    PubMed Central

    GELFAND, ROBERT; VERNET, DOLORES; BRUHN, KEVIN; VADGAMA, JAYDUTT; GONZALEZ-CADAVID, NESTOR F.

    2016-01-01

    Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0–2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells. PMID:27035792

  16. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    SciTech Connect

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. )

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  17. Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2

    PubMed Central

    Massari, Paola

    2015-01-01

    Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated that Neisseria gonorrhoeae, a Gram-negative pathogen responsible for the sexually transmitted infection gonorrhea, protects against exogenous induction of apoptosis in human cervical epithelial cells. However, induction of cell death by N. gonorrhoeae has also been reported in other cell types. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed that N. gonorrhoeae induces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early after N. gonorrhoeae stimulation, which was followed by a marked decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes and an overall increase in production of exosomes. Inhibition of cIAP2 in N. gonorrhoeae-stimulated epithelial cells resulted in increased cell death and interleukin-1β (IL-1β) production. Collectively these results indicate that N. gonorrhoeae stimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling. PMID:26077759

  18. Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2.

    PubMed

    Nudel, Kathleen; Massari, Paola; Genco, Caroline A

    2015-09-01

    Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated that Neisseria gonorrhoeae, a Gram-negative pathogen responsible for the sexually transmitted infection gonorrhea, protects against exogenous induction of apoptosis in human cervical epithelial cells. However, induction of cell death by N. gonorrhoeae has also been reported in other cell types. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed that N. gonorrhoeae induces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early after N. gonorrhoeae stimulation, which was followed by a marked decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes and an overall increase in production of exosomes. Inhibition of cIAP2 in N. gonorrhoeae-stimulated epithelial cells resulted in increased cell death and interleukin-1β (IL-1β) production. Collectively these results indicate that N. gonorrhoeae stimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling.

  19. Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function.

    PubMed

    Browne, James A; Yang, Rui; Song, Lingyun; Crawford, Gregory E; Leir, Shih-Hsing; Harris, Ann

    2014-12-01

    The epithelium lining the epididymis in the male reproductive tract maintains a luminal environment that promotes sperm cell maturation. This process is dependent on the coordinated expression of many genes that encode proteins with a role in epithelial transport. We previously generated genome-wide maps of open chromatin in primary human epididymis epithelial (HEE) cells to identify potential regulatory elements controlling coordinated gene expression in the epididymis epithelium. Subsequent in silico analysis identified transcription factor-binding sites (TFBS) that were over-represented in the HEE open chromatin, including the motif for paired box 2 (PAX2). PAX2 is a critical transcriptional regulator of urogenital tract development, which has been well studied in the kidney but is unexplored in the epididymis. Due to the limited lifespan of primary HEE cells in culture, we investigated the role of PAX2 in an immortalized HEE cell line (REP). First, REP cells were evaluated by DNase I digestion followed by high-throughput sequencing and the PAX2-binding motif was again identified as an over-represented TFBS within intergenic open chromatin, though on fewer chromosomes than in the primary HEE cells. To identify PAX2-target genes in REP cells, RNA-seq analysis was performed after siRNA-mediated depletion of PAX2 and compared with that with a non-targeting siRNA. In response to PAX2-repression, 3135 transcripts were differentially expressed (1333 up-regulated and 1802 down-regulated). Novel PAX2 targets included multiple genes encoding proteins with predicted functions in the epididymis epithelium.

  20. Pro-inflammatory responses of human bronchial epithelial cells to acute nitrogen dioxide exposure.

    PubMed

    Ayyagari, Vijayalakshmi N; Januszkiewicz, Adolph; Nath, Jayasree

    2004-04-15

    Nitrogen dioxide (NO2) is an environmental oxidant, known to be associated with lung epithelial injury. In the present study, cellular pro-inflammatory responses following exposure to a brief high concentration of NO2 (45 ppm) were assessed, using normal human bronchial epithelial (NHBE) cells as an in vitro model of inhalation injury. Generation and release of pro-inflammatory mediators such as nitric oxide (NO), IL-8, TNF-alpha, IFN-gamma and IL-1beta were assessed at different time intervals following NO2 exposure. Effects of a pre-existing inflammatory condition was tested by treating the NHBE cells with different inflammatory cytokines such as IFN-gamma, IL-8, TNF-alpha, IL-1beta, either alone or in combination, before exposing them to NO2. Immunofluorescence studies confirmed oxidant-induced formation of 3-nitrotyrosine in the NO2-exposed cells. A marked increase in the levels of nitrite (as an index of NO) and IL-8 were observed in the NO2-exposed cells, which were further enhanced in the presence of the cytokines. Effects of various NO inhibitors combined, with immunofluorescence and Western blotting data, indicated partial contribution of the nitric oxide synthases (NOSs) toward the observed increase in nitrite levels. Furthermore, a significant increase in IL-1beta and TNF-alpha generation was observed in the NO2-exposed cells. Although NO2 exposure alone did induce slight cytotoxicity (<12%), but presence of inflammatory cytokines such as TNF-alpha and IFN-gamma resulted in an increased cell death (28-36%). These results suggest a synergistic role of inflammatory mediators, particularly of NO and IL-8, in NO2-mediated early cellular changes. Our results also demonstrate an increased sensitivity of the cytokine-treated NHBE cells toward NO2, which may have significant functional implications in vivo.

  1. 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.

  2. Human metapneumovirus glycoprotein G disrupts mitochondrial signaling in airway epithelial cells.

    PubMed

    Bao, Xiaoyong; Kolli, Deepthi; Ren, Junping; Liu, Tianshuang; Garofalo, Roberto P; Casola, Antonella

    2013-01-01

    Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS (mitochondrial antiviral signaling protein), recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.

  3. Estradiol antagonism of glucocorticoid-induced GILZ expression in human uterine epithelial cells and murine uterus.

    PubMed

    Whirledge, Shannon; Cidlowski, John A

    2013-01-01

    Sex hormone signaling regulates a variety of functions in the uterine endometrium essential for embryo implantation and immunity. Epithelial cells of the uterine endometrium are the target of the coordinated actions of estradiol (E(2)) and progesterone. However, little information exists regarding the interplay of estrogens with glucocorticoids in this tissue. Using the human uterine epithelial cell line ECC1, E(2) was found to antagonize induction of the glucocorticoid-induced leucine zipper (GILZ) gene expression, which is associated with several of the immune-related functions of glucocorticoids. Interestingly, E(2) antagonizes glucocorticoid regulated nascent RNA GILZ expression within 1 h of hormone treatment. Repression of glucocorticoid-induced GILZ expression requires the estrogen receptor (ER), because both treatment with the ER-antagonist ICI 182,780 and small interfering RNA knockdown of ERα block E(2)'s ability to repress GILZ gene expression. Antagonism of glucocorticoid-induced GILZ expression may not be unique to ERα, as the ERβ agonist Liquiritigenin is also able to antagonize glucocorticoid signaling. Transcriptional regulation appears to be at the level of promoter binding. Both the glucocorticoid receptor and ERα are recruited to regions of the GILZ promoter containing glucocorticoid response elements and the transcriptional start site. Glucocorticoid receptor binding to these regions in the presence of dexamethasone decreases with E(2) treatment. GILZ gene expression was also found to be repressed in the whole mouse uterus treated with a combination of dexamethasone and E(2). Regulation of the antiinflammatory gene GILZ by glucocorticoids and E(2) suggests cross talk between the immune modulating functions of glucocorticoids and the reproductive actions of estradiol signaling.

  4. Apical uptake of choline and cationic drugs in epithelial cell lines derived from human placenta.

    PubMed

    Müller, J; Born, I; Neubert, R H; Brandsch, M

    2005-01-01

    Many cationic drugs are administered during pregnancy and might enter the fetal circulation by transplacental passage. This study was performed to characterize the apical uptake of choline and several cationic drugs at cultured epithelial cells of the human placenta. Total uptake of [3H]choline in BeWo cells was H(+)-independent and to 65% Na(+)-independent. Uptake rates into both cell lines were saturable with Michaelis-Menten constants (Kt) of 108 microM (BeWo) and 206 microM (JEG-3), respectively. Cationic drugs such as etilefrine, clonidine, ranitidine, diphenhydramine, imipramine and butylscopolamine strongly inhibited the [3H]choline uptake in BeWo cells and in JEG-3 cells, with Ki values ranging from 0.18 to 3.3 mM. In contrast, tetraethylammonium had only little inhibitory effect on [3H]choline uptake. Using high-performance capillary electrophoresis for quantitative analyses, uptake of etilefrine and diphenhydramine into JEG-3 or BeWo cells was measured. Diphenhydramine was transported into JEG-3 cells in a saturable manner with a Kt value of 0.75 mM. In the presence of sodium, diphenhydramine uptake at BeWo cells was inhibited to 69% by the addition of 50 mM choline chloride. Like choline uptake, total diphenhydramine uptake was to 68% Na(+)-independent in BeWo cells. We conclude that in addition to choline, several cationic drugs, in particular diphenhydramine, are taken up by placental epithelial cells from the maternal blood by carrier-mediated processes. Etilefrine, clonidine, ranitidine, diphenhydramine and butylscopolamine interact with the Na(+)-independent placental choline transport system.

  5. Expression of TRPC6 channels in human epithelial breast cancer cells

    PubMed Central

    Guilbert, Arnaud; Dhennin-Duthille, Isabelle; Hiani, Yassine EL; Haren, Nathalie; Khorsi, Hafida; Sevestre, Henri; Ahidouch, Ahmed; Ouadid-Ahidouch, Halima

    2008-01-01

    Background TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues. Methods Molecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration). Results A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPM7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours. Conclusion Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis. PMID:18452628

  6. In vitro cadmium effects on ECM gene expression in human bronchial epithelial cells.

    PubMed

    Baroni, Tiziano; Lilli, Cinzia; Bellucci, Catia; Luca, Giovanni; Mancuso, Francesca; Fallarino, Francesca; Falabella, Giulia; Arato, Iva; Calvitti, Mario; Marinucci, Lorella; Muzi, Giacomo; Dell'Omo, Marco; Gambelunghe, Angela; Bodo, Maria

    2015-03-01

    Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFβ/TGFβ receptor (TGFβR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, β1 and β5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and β3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFβ and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFβ/TGFβR signaling.

  7. 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.

  8. Calcium dependent and independent cytokine synthesis by air pollution particle-exposed human bronchial epithelial cells

    SciTech Connect

    Sakamoto, Noriho; Hayashi, Shizu; Gosselink, John; Ishii, Hiroshi; Ishimatsu, Yuji; Mukae, Hiroshi; Hogg, James C.; Eeden, Stephan F. van

    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 to 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.

  9. 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

  10. MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro.

    PubMed

    Fang, Shijing; Crews, Anne L; Chen, Wei; Park, Joungjoa; Yin, Qi; Ren, Xiu-Rong; Adler, Kenneth B

    2013-04-15

    Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

  11. MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

    PubMed Central

    Fang, Shijing; Crews, Anne L.; Chen, Wei; Park, Joungjoa; Yin, Qi; Ren, Xiu-Rong

    2013-01-01

    Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation. PMID:23377348

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

    PubMed

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

    2014-10-01

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

  13. Antagonistic effect of Candida albicans and IFNγ on E-cadherin expression and production by human primary gingival epithelial cells.

    PubMed

    Rouabhia, Mahmoud; Semlali, Abdelhabib; Audoy, Julie; Chmielewski, Witold

    2012-11-01

    Caused mainly by Candida albicans, oropharyngeal candidiasis is the most common oral complication associated with HIV disease worldwide. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral mucosa is the first line of defense in the form of a physical barrier against C. albicans invasion, and since epithelial cells are involved in anti-Candida innate immunity through different cytokines, we wanted to determine whether C. albicans alters E-cadherin expression and production, and whether interferon-γ (INFγ), a TH1 cytokine, is involved in the anti-Candida defense. Using primary human gingival epithelial cells, we demonstrated that C. albicans significantly decreased E-cadherin mRNA expression and protein production. This effect was basically obtained at later infective periods (24 and 48h). Interestingly, when IFNγ was added to C. albicans infected epithelial cell cultures, it prevented the side effect of C. albicans on E-cadherin mRNA expression and protein production and deposition. All together, these results suggested concomitant interactions between oral epithelial cells and IFNγ against C. albicans infection.

  14. Effects of the Probiotic Enterococcus faecium and Pathogenic Escherichia coli Strains in a Pig and Human Epithelial Intestinal Cell Model

    PubMed Central

    Lodemann, Ulrike; Strahlendorf, Julia; Schierack, Peter; Klingspor, Shanti; Aschenbach, Jörg R.

    2015-01-01

    The aim of this study has been to elucidate the effect of the probiotic Enterococcus faecium NCIMB 10415 on epithelial integrity in intestinal epithelial cells and whether pre- and coincubation with this strain can reproducibly prevent damage induced by enterotoxigenic (ETEC) and enteropathogenic Escherichia coli (EPEC). Porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells were incubated with bacterial strains and epithelial integrity was assessed by measuring transepithelial electrical resistance (TEER) and mannitol flux rates. E. faecium alone increased TEER of Caco-2 cells without affecting mannitol fluxes whereas the E. coli strains decreased TEER and concomitantly increased mannitol flux rates in both cell lines. Preincubation with E. faecium had no effect on the TEER decrease induced by E. coli in preliminary experiments. However, in a second set of experiments using a slightly different protocol, E. faecium ameliorated the TEER decrease induced by ETEC at 4 h in IPEC-J2 and at 2, 4, and 6 h in Caco-2 cells. We conclude that E. faecium positively affected epithelial integrity in monoinfected Caco-2 cells and could ameliorate the damage on TEER induced by an ETEC strain. Reproducibility of the results is, however, limited when experiments are performed with living bacteria over longer periods. PMID:25883829

  15. Epithelial Cell Secretions from the Human Female Reproductive Tract Inhibit Sexually Transmitted Pathogens and Candida albicans but not Lactobacillus

    PubMed Central

    Wira, CR; Ghosh, M; Smith, JM; Shen, L; Connor, RI; Sundstrom, P; Frechette, Gregory M.; Hill, EM; Fahey, JV

    2011-01-01

    Female reproductive tract (FRT) epithelial cells protect against potential pathogens and sexually transmitted infections. The purpose of this study was to determine if epithelial cells from the upper FRT secrete antimicrobials that inhibit reproductive tract pathogens which threaten women's health. Apical secretions from primary cultures of Fallopian tube, uterine, cervical and ectocervical epithelial cells were incubated with Neisseria gonorrhoeae, Candida albicans (yeast and hyphal forms), HIV-1, and Lactobacillus crispatus, prior to being tested for their ability to grow and/or infect target cells. Epithelial cell secretions from the upper FRT inhibit N. gonorrhoeae and both forms of Candida, as well as reduce HIV-1 (R5) infection of target cells. In contrast, none had an inhibitory effect on L. crispatus. Cytokines and chemokines analysis in uterine secretions revealed several molecules that could account for pathogen inhibition. These findings provide definitive evidence for the critical role of epithelial cells in protecting the FRT from infections, without comprising the beneficial presence of L. crispatus, which is part of the normal vaginal microflora of humans. PMID:21048705

  16. Campylobacter jejuni Induces Secretion of Proinflammatory Chemokines from Human Intestinal Epithelial Cells

    DTIC Science & Technology

    2005-07-01

    Gudis, T. Tsukui, and C. Sakamoto. 2003. Monocyte chemoat- tractant protein 1 (MCP-1) released from Helicobacter pylori stimulated gastric epithelial...S. Day. 2004. Gastric epithelial cell CXC chemokine secretion following Helicobacter pylori infection in vitro. J. Gastroenterol. Hepatol. 19:982...K. Kashima, and J. Imanishi. 1998. Chemokines in the gastric mucosa in Helicobacter pylori infection. Gut 42:609–617. 15. Yang, S. K., L. Eckmann, A

  17. Pathogen and host differences in bacterial adherence to human buccal epithelial cells in a northeast Brazilian community.

    PubMed Central

    Walser, B L; Newman, R D; Lima, A A; Guerrant, R L

    1992-01-01

    The adherence of several strains of Escherichia coli to human buccal epithelial cells was studied, using cells obtained from five groups: healthy adults, healthy children, children with acute diarrhea, children with persistent diarrhea associated with cryptosporidial parasites, and children with noncryptosporidial persistent diarrhea. All groups lived or worked in an urban slum in northeastern Brazil. Samples of buccal epithelial cells from subjects in each of these groups were incubated with wild-type E. coli K-12 (strain C600), the enteroaggregative E. coli strains 17-2 and PDAS 30-5, CFA/II-positive E. coli 1392+ and its plasmid-cured derivative 1392-, and hydrophobic E. coli 132-3. Samples were evaluated microscopically to determine background contamination and the percentage of cells with more than 15% of their surface area obscured by adherent bacteria after incubation and washing. The assay was tested under field conditions and was shown to produce reliable and consistent results. Both enteroaggregative strains of E. coli were shown to adhere to a significantly higher percentage of all groups of human buccal epithelial cells than any of the other tested strains. In addition, buccal epithelial cells from children with nonparasitic persistent diarrhea showed substantially more bacterial adherence in both the native state and with all tested strains of E. coli than did cells from children with persistent cryptosporidial diarrhea or acute diarrhea or from healthy controls. This study provides evidence that enteroaggregative strains of E. coli demonstrate increased adherence to human buccal epithelial cells (as well as to cultured HEp-2 cells) and that buccal epithelial cells from children with noncryptosporidial persistent diarrhea appear to be more susceptible to bacterial adherence and colonization than buccal epithelial cells from control groups. These findings suggest that host differences as well as pathogen differences are important in the pathogenesis of

  18. Human Airway Epithelial Cell Responses to Single Walled Carbon Nanotube Exposure: Nanorope-Residual Body Formation

    SciTech Connect

    Panessa-Warren, Barbara J.; Warren, John B.; Kisslinger, Kim; Crosson, Kenya; Maye, Mathew M.

    2012-11-01

    This investigation examines the 'first contact responses' of in vitro human epithelial airway cells exposed to unrefined single walled carbon nanotubes (SWCNTs) [containing metal catalyst, carbon black, amorphous carbon, graphitic shells, and SWCNTs], and refined acid/peroxide cleaned and cut SWCNTs at low and high dose exposures (0.16 ug/L and 1.60 ug/L) for 2, 3 and 3.5 hours. FTIR, X-ray compositional analysis, morphological TEM analysis and UV-Vis were used to physicochemically characterize the SWCNTs in this study. Following SWCNT exposure to human lung NCI-H292 epithelial monolayers, the airway cells were prepared for light microscopy vital staining, or fixed in glutaraldehyde for SEM/TEM imaging to determine SWCNT binding, uptake, intracellular processing and organellar/SWCNT fate within the exposure period. At 2 hr exposures to both unrefined Carbolex, and refined SWCNTs (at both high and low doses), there were no increases in lung cell necrosis compared to controls. However high dose, 3 hr exposures to unrefined Carbolex material produced severe cell damage (apical and basal plasma membrane holes, decreased mitochondria, numerous intracellular vesicles containing nanomaterial and membrane fragments) and increased cell necrosis. The refined SWCNTs exposed for 3 hr at low dose produced no increase in cell death, although high dose exposure produced significant cell death. By TEM, Acid/peroxide cleaned SWCNT 3 hr exposures at high and low doses, revealed SWCNTs attachment to cell surface mucin, and SWCNT uptake into the cells during membrane recycling. Membranes and SWCNTs were seen within cytoplasmic lamellar body-type vesicles, where vesicular contents were bio-degraded, eventually forming long SWCNT-nanoropes, which were subsequently released into the cytoplasm as clusters of attached nanoropes, as the vesicle membranes fragmented. These Nanorope-Residual Bodies did not cause damage to the surrounding organelles or cytoplasm, and seemed very stabile in the

  19. Vitamin D Activation and Function in Human Corneal Epithelial Cells During TLR-Induced Inflammation

    PubMed Central

    Reins, Rose Yvonne; Baidouri, Hasna; McDermott, Alison Marie

    2015-01-01

    Purpose Vitamin D is recognized to be an important modulator of the immune system. In the eye, studies have shown that deficiencies and genetic differences in vitamin D–related genes have a significant impact on the development of various ocular diseases. Our current study examines the ability of human corneal epithelial cells (HCEC) to activate vitamin D and the effect of vitamin D treatment on antimicrobial peptide production and cytokine modulation during inflammation, with the ultimate goal of using vitamin D therapeutically for corneal inflammation. Methods Human corneal epithelial cells were treated with 10−7M vitamin D3 (D3) or 25-hydroxyvitamin D3 (25D3) for 24 hours and 1,25-dihydroxyvitamin D3 (1,25D3) detected by immunoassay. Human cathelicidin (LL-37) expression was examined by RT-PCR, immunoblot, and immunostaining following 1,25D3 treatment and antimicrobial activity of 1,25D3-treated cells was determined. Cells were stimulated with TLR3 agonist polyinosinic-polycytidylic acid (Poly[I:C]) for 24 hours and cytokine levels measured by RT-PCR, ELISA, and Luminex. Immunostaining determined expression of vitamin D receptor (VDR) and retinoic acid inducible gene-1 receptor (RIG-1) as well as NF-κB nuclear translocation. Results When treated with inactive vitamin D metabolites, HCEC produced active 1,25D3, leading to enhanced expression of the antimicrobial peptide, LL-37, dependent on VDR. 1,25-D3 decreased the expression of proinflammatory cytokines (IL-1β, IL-6, TNFα, and CCL20) and MMP-9 induced by Poly(I:C) as well as pattern recognition receptor expression (TLR3, RIG-1, MDA5). However, early activation of NF-κB was not affected. Conclusions These studies demonstrate the protective ability of vitamin D to attenuate proinflammatory mediators while increasing antimicrobial peptides and antipseudomonas activity in corneal cells, and further our knowledge on the immunomodulatory functions of the hormone. PMID:26641549

  20. 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

  1. Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis.

    PubMed

    Teoh-Fitzgerald, M L; Fitzgerald, M P; Zhong, W; Askeland, R W; Domann, F E

    2014-01-16

    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

  2. A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain.

    PubMed

    Moe, Morten C; Kolberg, Rebecca S; Sandberg, Cecilie; Vik-Mo, Einar; Olstorn, Havard; Varghese, Mercy; Langmoen, Iver A; Nicolaissen, Bjørn

    2009-01-01

    Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes (n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy (n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10-30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-beta receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human

  3. Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways

    PubMed Central

    Maestre-Batlle, Danay; Pena, Olga M.; Hirota, Jeremy A.; Gunawan, Evelyn; Rider, Christopher F.; Sutherland, Darren; Alexis, Neil E.; Carlsten, Chris

    2017-01-01

    Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. PMID:28165060

  4. Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)*

    PubMed Central

    Tsaktanis, Thanos; Kremling, Heidi; Pavšič, Miha; von Stackelberg, Ricarda; Mack, Brigitte; Fukumori, Akio; Steiner, Harald; Vielmuth, Franziska; Spindler, Volker; Huang, Zhe; Jakubowski, Jasmine; Stoecklein, Nikolas H.; Luxenburger, Elke; Lauber, Kirsten; Lenarčič, Brigita; Gires, Olivier

    2015-01-01

    Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable. PMID:26292218

  5. Role of connexin 43 in cadmium-induced proliferation of human prostate epithelial cells.

    PubMed

    Liu, Qingping; Ji, Xiaoli; Ge, Zehe; Diao, Haipeng; Chang, Xiuli; Wang, Lihua; Wu, Qing

    2017-02-08

    Connexins (Cxs), the subunits of gap junction channels, are involved in many physiological processes. Aberrant control of Cxs and gap junction intercellular communication may contribute to many diseases, including the promotion of cancer. Cd exposure is associated with increased risk of human prostate cancer and benign prostatic hyperplasia. The roles of Cxs in the effects of Cd on the prostate have, however, not been reported previously. In this study, the human prostate epithelial cell line RWPE-1 was exposed to Cd. A low dose of Cd stimulated cell proliferation along with a lower degree of gap junction intercellular communication and an elevated level of the protein Cx43. Cd exposure increased the levels of intracellular Ca(2+) and phosphorylated Cx43 at the Ser368 site. Knockdown of Cx43 using siRNA blocked Cd-induced proliferation and interfered with the Cd-induced changes in the protein levels of cyclin D1, cyclin B1, p27(Kip1) (p27) and p21(Waf1/Cip1) (p21). The increase in Cx43 expression induced by Cd was presumably mediated by the androgen receptor, because it was abolished upon treatment with the androgen receptor antagonist, flutamide. Thus, a low dose of Cd promotes cell proliferation in RWPE-1, possibly mediated by Cx43 expression through an effect on cell cycle-associated proteins. Cx43 might be a target for prostatic diseases associated with Cd exposure. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Proteomic Analysis of Primary Human Airway Epithelial Cells Exposed to the Respiratory Toxicant Diacetyl.

    PubMed

    Foster, Matthew W; Gwinn, William M; Kelly, Francine L; Brass, David M; Valente, Ashlee M; Moseley, M Arthur; Thompson, J Will; Morgan, Daniel L; Palmer, Scott M

    2017-02-03

    Occupational exposures to the diketone flavoring agent, diacetyl, have been associated with bronchiolitis obliterans, a rare condition of airway fibrosis. Model studies in rodents have suggested that the airway epithelium is a major site of diacetyl toxicity, but the effects of diacetyl exposure upon the human airway epithelium are poorly characterized. Here we performed quantitative LC-MS/MS-based proteomics to study the effects of repeated diacetyl vapor exposures on 3D organotypic cultures of human primary tracheobronchial epithelial cells. Using a label-free approach, we quantified approximately 3400 proteins and 5700 phosphopeptides in cell lysates across four independent donors. Altered expression of proteins and phosphopeptides were suggestive of loss of cilia and increased squamous differentiation in diacetyl-exposed cells. These phenomena were confirmed by immunofluorescence staining of culture cross sections. Hyperphosphorylation and cross-linking of basal cell keratins were also observed in diacetyl-treated cells, and we used parallel reaction monitoring to confidently localize and quantify previously uncharacterized sites of phosphorylation in keratin 6. Collectively, these data identify numerous molecular changes in the epithelium that may be important to the pathogenesis of flavoring-induced bronchiolitis obliterans. More generally, this study highlights the utility of quantitative proteomics for the study of in vitro models of airway injury and disease.

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

    PubMed

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

    2014-12-21

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

  8. Bdellovibrio bacteriovorus Inhibits Staphylococcus aureus Biofilm Formation and Invasion into Human Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Monnappa, Ajay K.; Dwidar, Mohammed; Seo, Jeong Kon; Hur, Jin-Hoe; Mitchell, Robert J.

    2014-01-01

    Bdellovibrio bacteriovorus HD100 is a predatory bacterium that attacks many Gram-negative human pathogens. A serious drawback of this strain, however, is its ineffectiveness against Gram-positive strains, such as the human pathogen Staphylococcus aureus. Here we demonstrate that the extracellular proteases produced by a host-independent B. bacteriovorus (HIB) effectively degrade/inhibit the formation of S. aureus biofilms and reduce its virulence. A 10% addition of HIB supernatant caused a 75% or greater reduction in S. aureus biofilm formation as well as 75% dispersal of pre-formed biofilms. LC-MS-MS analyses identified various B. bacteriovorus proteases within the supernatant, including the serine proteases Bd2269 and Bd2321. Tests with AEBSF confirmed that serine proteases were active in the supernatant and that they impacted S. aureus biofilm formation. The supernatant also possessed a slight DNAse activity. Furthermore, treatment of planktonic S. aureus with the supernatant diminished its ability to invade MCF-10a epithelial cells by 5-fold but did not affect the MCF-10a viability. In conclusion, this study illustrates the hitherto unknown ability of B. bacteriovorus to disperse Gram-positive pathogenic biofilms and mitigate their virulence.

  9. Role of mitochondrial permeability transition in human renal tubular epithelial cell death induced by aristolochic acid

    SciTech Connect

    Qi Xinming; Cai Yan; Gong Likun; Liu Linlin; Chen Fangping; Xiao Ying; Wu Xiongfei; Li Yan; Xue Xiang |; Ren Jin . E-mail: cdser_simm@mail.shcnc.ac.cn

    2007-07-01

    Aristolochic acid (AA), a natural nephrotoxin and carcinogen, can induce a progressive tubulointerstitial nephropathy. However, the mechanism by which AA causes renal injury remains largely unknown. Here we reported that the mitochondrial permeability transition (MPT) plays an important role in the renal injury induced by aristolochic acid I (AAI). We found that in the presence of Ca{sup 2+}, AAI caused mitochondrial swelling, leakage of Ca{sup 2+}, membrane depolarization, and release of cytochrome c in isolated kidney mitochondria. These alterations were suppressed by cyclosporin A (CsA), an agent known to inhibit MPT. Culture of HK-2 cell, a human renal tubular epithelial cell line for 24 h with AAI caused a decrease in cellular ATP, mitochondrial membrane depolarization, cytochrome c release, and increase of caspase 3 activity. These toxic effects of AAI were attenuated by CsA and bongkrekic acid (BA), another specific MPT inhibitor. Furthermore, AAI greatly inhibited the activity of mitochondrial adenine nucleotide translocator (ANT) in isolated mitochondria. We suggested that ANT may mediate, at least in part, the AAI-induced MPT. Taken together, these results suggested that MPT plays a critical role in the pathogenesis of HK-2 cell injury induced by AAI and implied that MPT might contribute to human nephrotoxicity of aristolochic acid.

  10. 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.

  11. 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.

  12. Human IgA inhibits adherence of Acanthamoeba polyphaga to epithelial cells and contact lenses.

    PubMed

    Campos-Rodríguez, Rafael; Oliver-Aguillón, Gabriela; Vega-Pérez, Luz M; Jarillo-Luna, Adriana; Hernández-Martínez, Dolores; Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A; Rivera-Aguilar, Víctor; González-Robles, Arturo

    2004-09-01

    Specific anti-Acanthamoeba IgA antibodies have been detected in the serum and tears of patients and healthy individuals. However, the role of human secretory IgA antibodies in inhibiting the adherence of Acanthamoeba had not been previously investigated. Therefore, the purpose of this study was to purify secretory IgA from human colostrum and analyze its effect on the adherence of Acanthamoeba trophozoites to contact lenses and Madin-Darby canine kidney (MDCK) cells. IgA antibodies to Acanthamoeba polyphaga in colostrum of healthy women as well as in saliva and serum of healthy subjects were analyzed by ELISA and Western blot analysis. In serum, saliva, and colostrum, we detected IgA antibodies that recognized several antigens of A. polyphaga. In addition, colostrum and IgA antibodies purified from it inhibited adherence of A. polyphaga trophozoites to contact lenses and MDCK cells. These results suggest that IgA antibodies may participate in the resistance to the amoebic infection, probably by inhibiting the adherence of the trophozoites to contact lenses and corneal epithelial cells.

  13. Microsatellite instability in human mammary epithelial cells transformed by heavy ions

    NASA Astrophysics Data System (ADS)

    Yanada, S.; Yang, T. C.; George, K.; Okayasu, R.; Ando, K.; Tsujii, H.

    1998-11-01

    We analyzed DNA and proteins obtained from normal and transformed human mammary epithelial cells for studying the neoplastic transformation by high-LET irradiation in vitro. We also examined microsatellite instability in human mammary cells transformed to various stages of carcinogenesis, such as normal, growth variant and tumorigenic, using microsatellite marker D5S177 on the chromosome 5 and CY17 on the Chromosome 10. Microsatellite instabilities were detected in the tumorigenic stage. These results suggest that microsatellite instability may play a role in the progression of tumorigenecity. The cause of the genomic instability has been suggested as abnormalities of DNA-repair systems which may be due to one of the three reasons: 1) alterations of cell cycle regulating genes. 2) mutations in any of the DNA mismatch repair genes, 3) mutation in any of the DNA strand breaks repair genes. No abnormality of these genes and encoded proteins, however was found in the present studies. These studies thus suggest that the microsatellite instability is induced by an alternative mechanism.

  14. Development and Application of Human Renal Proximal Tubule Epithelial Cells for Assessment of Compound Toxicity

    PubMed Central

    Li, Shuaizhang; Zhao, Jinghua; Huang, Ruili; Steiner, Toni; Bourner, Maureen; Mitchell, Michael; Thompson, David C.; Zhao, Bin; Xia, Menghang

    2017-01-01

    Kidney toxicity is a major problem both in drug development and clinical settings. It is difficult to predict nephrotoxicity in part because of the lack of appropriate in vitro cell models, limited endpoints, and the observation that the activity of membrane transporters which plays important roles in nephrotoxicity by affecting the pharmacokinetic profile of drugs is often not taken into account. We developed a new cell model using pseudo-immortalized human primary renal proximal tubule epithelial cells. This cell line (SA7K) was characterized by the presence of proximal tubule cell markers as well as several functional properties, including transporter activity and response to a few well-characterized nephrotoxicants. We subsequently evaluated a group of potential nephrotoxic compounds in SA7K cells and compared them to a commonly used human immortalized kidney cell line (HK-2). Cells were treated with test compounds and three endpoints were analyzed, including cell viability, apoptosis and mitochondrial membrane potential. The results showed that most of the known nephrotoxic compounds could be detected in one or more of these endpoints. There were sensitivity differences in response to several of the chemicals between HK-2 and SA7K cells, which may relate to differences in expressions of key transporters or other components of nephrotoxicity pathways. Our data suggest that SA7K cells appear as promising for the early detection of renal toxicants.

  15. Melissa Officinalis L. Extracts Protect Human Retinal Pigment Epithelial Cells against Oxidative Stress-Induced Apoptosis

    PubMed Central

    Jeung, In Cheul; Jee, Donghyun; Rho, Chang-Rae; Kang, Seungbum

    2016-01-01

    Background: We evaluated the protective effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells (ARPE-19 cells). Methods: ARPE-19 cells were incubated with ALS-L1023 for 24 h and then treated with hydrogen peroxide (H2O2). Oxidative stress-induced apoptosis and intracellular generation of reactive oxygen species (ROS) were assessed by flow cytometry. Caspase-3/7 activation and cleaved poly ADP-ribose polymerase (PARP) were measured to investigate the protective role of ALS-L1023 against apoptosis. The protective effect of ALS-L1023 against oxidative stress through activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) was evaluated by Western blot analysis. Results: ALS-L1023 clearly reduced H2O2-induced cell apoptosis and intracellular production of ROS. H2O2-induced oxidative stress increased caspase-3/7 activity and apoptotic PARP cleavage, which were significantly inhibited by ALS-L1023. Activation of the PI3K/Akt pathway was associated with the protective effect of ALS-L1023 on ARPE-19 cells. Conclusions: ALS-L1023 protected human RPE cells against oxidative damage. This suggests that ALS-L1023 has therapeutic potential for the prevention of dry age-related macular degeneration. PMID:26941573

  16. The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells.

    PubMed Central

    Rufo, P A; Jiang, L; Moe, S J; Brugnara, C; Alper, S L; Lencer, W I

    1996-01-01

    Clotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals. PMID:8903326

  17. Human intestinal epithelial cells produce proinflammatory cytokines in response to infection in a SCID mouse-human intestinal xenograft model of amebiasis.

    PubMed Central

    Seydel, K B; Li, E; Swanson, P E; Stanley, S L

    1997-01-01

    The protozoan parasite Entamoeba histolytica causes amebic dysentery and amebic liver abscess, diseases associated with significant morbidity and mortality worldwide. E. histolytica infection appears to involve the initial attachment of amebic trophozoites to intestinal epithelial cells, followed by lysis of these cells and subsequent invasion into the submucosa. A recent in vitro study (L. Eckmann, S. L. Reed, J. R. Smith, and M. F. Kagnoff, J. Clin. Invest. 96:1269-1279, 1995) demonstrated that incubation of E. histolytica trophozoites with epithelial cell lines results in epithelial cell production of inflammatory cytokines, including interleukin-1 (IL-1) and IL-8, suggesting that intestinal epithelial cell production of cytokines might play a role in the inflammatory response and tissue damage seen in intestinal amebiasis. To determine whether intestinal epithelial cell production of IL-1 and IL-8 occurs in response to E. histolytica infection in vivo and as an approach to studying the specific interactions between amebic trophozoites and human intestine, we used a SCID mouse-human intestinal xenograft (SCID-HU-INT) model of disease, where human intestinal xenografts were infected with virulent E. histolytica trophozoites. Infection of xenografts with E. histolytica trophozoites resulted in extensive tissue damage, which was associated with the development of an early inflammatory response composed primarily of neutrophils. Using oligonucleotide primers that specifically amplify human IL-1beta and IL-8, we could demonstrate by reverse transcription PCR that mRNA for both IL-1beta and IL-8 is produced by human intestinal xenografts in response to amebic infection. The increase in human intestinal IL-1beta and IL-8 in response to invasive amebiasis was confirmed by enzyme-linked immunosorbent assays specific for human IL-1beta and IL-8. Using immunohistochemistry, we confirmed that human intestinal epithelial cells were the source of IL-8 in infected xenografts

  18. Distinct Effects of EGFR Ligands on Human Mammary Epithelial Cell Differentiation

    PubMed Central

    Mukhopadhyay, Chandrani; Zhao, Xiangshan; Maroni, Dulce; Band, Vimla; Naramura, Mayumi

    2013-01-01

    Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5+K19- hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5+K19- hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5+K19- hMEC differentiation. The present data validate the utility of the K5+K19- hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation. PMID:24124521

  19. Distinct effects of EGFR ligands on human mammary epithelial cell differentiation.

    PubMed

    Mukhopadhyay, Chandrani; Zhao, Xiangshan; Maroni, Dulce; Band, Vimla; Naramura, Mayumi

    2013-01-01

    Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5(+)K19(-) hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5(+)K19(-) hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5(+)K19(-) hMEC differentiation. The present data validate the utility of the K5(+)K19(-) hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation.

  20. Expression and secretion of immunoglobulin alpha heavy chain with diverse VDJ recombinations by human epithelial cancer cells.

    PubMed

    Zheng, Hui; Li, Ming; Ren, Wei; Zeng, Liang; Liu, Hai-dan; Hu, Duosha; Deng, Xiyun; Tang, Min; Shi, Ying; Gong, Jianping; Cao, Ya

    2007-03-01

    Generally, only B lymphocytes express immunoglobulin. Recently, we found the expression of Ig alpha heavy chain in human epithelial cancer cells unexpectedly. We first detected Ig VDJ-Calpha and Ialpha-Calpha transcripts in multiple cancer cell lines. Further, the configuration of the Ig heavy chain genomic locus was analyzed in human cancer cells. We found that cancer cells have the recombination VDJ region, but bear Ig Salpha region in germline configuration, which is different from Ig expression pattern in B cells. And human epithelial cancers possess the essential effectors including RAG-1 and RAG-2, but not activation induced cytidine deaminase (AID) protein. These provide further proofs for Ig alpha expression. In addition, we found that human cancer cells not only express the protein of Ig alpha chain, but also secrete the protein in secretory IgA (SIgA) pattern. Importantly, diverse CDR3 recombinations were found in human cancer cells of different epithelial origin. Since IgA is the key immunoglobulin which contributes to local immunity of mucous membrane, the aberrant expression of Ig alpha heavy chain might increase our further comprehension to development and immunity of cancers.

  1. MICAL2 is a novel human cancer gene controlling mesenchymal to epithelial transition involved in cancer growth and invasion.

    PubMed

    Mariotti, Sara; Barravecchia, Ivana; Vindigni, Carla; Pucci, Angela; Balsamo, Michele; Libro, Rosaliana; Senchenko, Vera; Dmitriev, Alexey; Jacchetti, Emanuela; Cecchini, Marco; Roviello, Franco; Lai, Michele; Broccoli, Vania; Andreazzoli, Massimiliano; Mazzanti, Chiara M; Angeloni, Debora

    2016-01-12

    The MICAL (Molecules Interacting with CasL) proteins catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/undifferentiated, primary human epithelial cancers (gastric and renal). Immunohistochemistry showed MICAL2-positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was 'on' in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, and was turned 'off' upon homing at metastatic sites. In vitro, MICAL2 knock-down resulted in mesenchymal to epithelial transition, reduction of viability, and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. Altogether our data indicate that MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy.

  2. MICAL2 is a novel human cancer gene controlling mesenchymal to epithelial transition involved in cancer growth and invasion

    PubMed Central

    Vindigni, Carla; Pucci, Angela; Balsamo, Michele; Libro, Rosaliana; Senchenko, Vera; Dmitriev, Alexey; Jacchetti, Emanuela; Cecchini, Marco; Roviello, Franco; Lai, Michele; Broccoli, Vania; Andreazzoli, Massimiliano; Mazzanti, Chiara M.; Angeloni, Debora

    2016-01-01

    The MICAL (Molecules Interacting with CasL) proteins catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/undifferentiated, primary human epithelial cancers (gastric and renal). Immunohistochemistry showed MICAL2-positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was 'on' in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, and was turned 'off' upon homing at metastatic sites. In vitro, MICAL2 knock-down resulted in mesenchymal to epithelial transition, reduction of viability, and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. Altogether our data indicate that MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy. PMID:26689989

  3. 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.

  4. 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

  5. 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

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

    PubMed Central

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

    2016-01-01

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

  7. Molecular Expression and Functional Evidence of a Drug Efflux Pump (BCRP) in Human Corneal Epithelial Cells

    PubMed Central

    Karla, Pradeep K.; Earla, Ravinder; Boddu, Sagar H.; Johnston, Thomas P.; Pal, Dhananjay; Mitra, Ashim

    2015-01-01

    Purpose Breast Cancer Resistance Protein (BCRP) belongs to the family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify the presence and possible role of BCRP as a bar-rier for ocular drug resistance. Methods Transfected human corneal epithelial cells (SV40-HCEC) were selected as an in vitro model for corneal epithelium with MDCKII-BCRP as positive control. [3H]-Mitoxantrone ([3H]-MTX), which is a proven substrate for organic anion transporter like BCRP, was selected as a model drug for functional expression studies. Fumetremorgin C (FTC), a known specific inhibitor for BCRP and GF120918, an inhibitor for BCRP and P-gp, were added to inhibit BCRP-mediated efflux. PGP-4008, a specific inhibitor of P-gp was used to delineate the contribution of P-gp. The mRNA extracted from cells was used for RT-PCR analysis and gene expression. Membrane fractions of SV40-HCEC and MDCKII-BCRP were used for immunoprecipitation followed by Western blot analysis. Results Efflux was inhibited significantly in the presence of FTC and GF120918. Dose-dependent inhibition of efflux by BCRP was noticed in SV40-HCEC and MDCKII-BCRP in the presence of FTC and GF120918, and the efflux was ATP-dependent. The metabolic inhibitor, 2,4-DNP, significantly inhibited efflux. No pH-dependent efflux was noticed except at pH 5.5. RT-PCR analysis indicated a unique and distinct band at ~429 bp, corresponding to BCRP in SV40-HCEC and MDCKII-BCRP cells. Western Blot analysis indicated a specific band at ~70 kDa in the membrane fraction of SV40-HCEC and MDCKII-BCRP cells. Conclusions We have demonstrated the expression of BCRP in human corneal epithelial cells and, for the first time, demonstrated its functional activity leading to drug efflux. RT-PCR and Western blot analysis further confirmed this finding. PMID:19172464

  8. Alisertib, an Aurora kinase A inhibitor, induces apoptosis and autophagy but inhibits epithelial to mesenchymal transition in human epithelial ovarian cancer cells.

    PubMed

    Ding, Yong-Hui; Zhou, Zhi-Wei; Ha, Chun-Fang; Zhang, Xue-Yu; Pan, Shu-Ting; He, Zhi-Xu; Edelman, Jeffrey L; Wang, Dong; Yang, Yin-Xue; Zhang, Xueji; Duan, Wei; Yang, Tianxin; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS), a selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT), and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS had potent growth-inhibitory, proapoptotic, proautophagic, and EMT-inhibitory effects on SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5'-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer cells

  9. Alisertib, an Aurora kinase A inhibitor, induces apoptosis and autophagy but inhibits epithelial to mesenchymal transition in human epithelial ovarian cancer cells

    PubMed Central

    Ding, Yong-Hui; Zhou, Zhi-Wei; Ha, Chun-Fang; Zhang, Xue-Yu; Pan, Shu-Ting; He, Zhi-Xu; Edelman, Jeffrey L; Wang, Dong; Yang, Yin-Xue; Zhang, Xueji; Duan, Wei; Yang, Tianxin; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS), a selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT), and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS had potent growth-inhibitory, proapoptotic, proautophagic, and EMT-inhibitory effects on SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5′-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer

  10. Epithelial membrane protein 2 (EMP2) deficiency alters placental angiogenesis, mimicking features of human placental insufficiency.

    PubMed

    Williams, Carmen J; Chu, Alison; Jefferson, Wendy N; Casero, David; Sudhakar, Deepthi; Khurana, Nevil; Hogue, Claire P; Aryasomayajula, Chinmayi; Patel, Priya; Sullivan, Peggy; Padilla-Banks, Elizabeth; Mohandessi, Shabnam; Janzen, Carla; Wadehra, Madhuri

    2017-03-14

    Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulate placental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to test the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2(-/-) ) were generated. Emp2(-/-) females are fertile but have reduced litter sizes when carrying Emp2(-/-) but not Emp2(+/-) fetuses. Placentas of Emp2(-/-) fetuses exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Placentas from Emp2(-/-) fetuses had increased total HIF-1α expression in large part through an increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by placental insufficiency resulting in intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1α expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in mice and humans and suggest it may be a new biomarker for placental insufficiency.

  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. Cadmium induces p53-dependent apoptosis in human prostate epithelial cells.

    PubMed

    Aimola, Pierpaolo; Carmignani, Marco; Volpe, Anna Rita; Di Benedetto, Altomare; Claudio, Luigi; Waalkes, Michael P; van Bokhoven, Adrie; Tokar, Erik J; Claudio, Pier Paolo

    2012-01-01

    Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl(2) and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl(2) concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.

  13. Effects of luminal thymol on epithelial transport in human and rat colon.

    PubMed

    Kaji, Izumi; Karaki, Shin-ichiro; Kuwahara, Atsukazu

    2011-06-01

    Gut lumen is continually exposed to a great variety of agents, including noxious compounds. Chemical receptors that detect the luminal environment are thought to play an important role as sensors and to modulate gastrointestinal functions. Recently, it has been reported that odorant receptors (ORs) are expressed in the small intestinal mucosa and that odorants stimulate serotonin secretion. However, ion transport in the responses to odorants has rarely been discussed, particularly in relation to the large intestine. In the present study, we examined the effects of the OR ligand thymol on ion transport in human and rat colonic epithelia using an Ussing chamber. In the mucosal-submucosal preparations, the mucosal addition of thymol evoked anion secretion concentration dependently. In addition, dextran (4 kDa) permeability was enhanced by the mucosal treatment with thymol. The response to thymol was not affected by tetrodotoxin (TTX) or piroxicam treatments in human or rat colon. Thymol-evoked electrogenic anion secretion was abolished under Ca(2+)-free conditions or mucosal treatment with transient receptor potential (TRP) A1 blocker (HC-030031). Pretreatment of thymol did not affect electrical field stimulation-evoked anion secretion but significantly attenuated short-chain fatty acid-evoked secretion in a concentration-dependent manner. OR1G1 and TRPA1 expression was investigated in isolated colonic mucosa by RT-PCR. The present results provide evidence that the OR ligand thymol modulates epithelial permeability and electrogenic anion secretion in human and rat colon. The anion secretion by luminal thymol is most likely mediated by direct activation of TRPA1 channel. We suggest that the sensing and responding to odorants in the colon also plays a role in maintaining intestinal homeostasis.

  14. Metabolism of aflatoxin B1 by normal human bronchial epithelial cells.

    PubMed

    Van Vleet, T R; Klein, P J; Coulombe, R A

    2001-08-10

    Aflatoxin B, (AFB1) is a potent hepatocarcinogen in animal models and a suspected carcinogen in humans. High concentrations of AFB, have been found in respirable grain dusts, and may therefore be a risk factor for human lung cancer in certain occupations. To study the potential for AFB, activation in human lung, cytochrome P-450 (CYP)-mediated activation and glutathione S-transferase (GST)-mediated detoxification of AFB1 were examined in cultured normal human bronchial epithelial (NHBE) cells. Cells were exposed to 0. 15 microM or 1.5 microM AFB, for 48 h and media was collected for metabolite analysis by high-performance liquid chromatography (HPLC). At 0. 15 microM, AFB1 was metabolized only to the detoxified metabolite aflatoxin Q1 (AFQ1). At 1.5 microM AFB1, both aflatoxin M1 (AFM1), and AFQ1 were produced. Cells pretreated with 50 degrees M 3-methylcholanthrene (3MC), a CYP 1A inducer, for 72 h prior to 0.15 microM AFB1, produced the activated AFB1 8,9-epoxide (AFBO). Similarly, microsomes prepared from 3MC-pretreated cells formed AFBO, but microsomes from noninduced cells did not. While AFB1-DNA adducts were not detected at low AFB1 concentrations in untreated NHBE, 3MC induction caused the production of AFB1-DNA adducts at 0.015 and 0.15 microM AFB1. Western immunoblots showed that the primary CYP isoforms responsible for AFB1 activation in the liver, 1A and 3A4, to be constitutively expressed in NHBE cells. Expression of CYP 1A was significantly increased in 3MC-pretreated cells, while CYP 3A4 expression increased slightly, but not to the extent of the 1A isoforms. The principal AFBO detoxifying enzyme, glutathione S-transferase (GST), was constitutively expressed in NHBE cells, and was increased approximately twofold by 3MC pretreatment. Cytosolic fractions from neither control nor 3MC-induced NHBE had measurable AFBO conjugating activity, indicating that these cells may lack AFB1-relevant GST activity. From these data, it appears that NHBE cells activate