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Sample records for caco-2 intestinal epithelial

  1. Bioavailability of genistein, daidzein, and their glycosides in intestinal epithelial Caco-2 cells

    NARCIS (Netherlands)

    Steensma, A.; Noteborn, H.P.J.M.; Jagt, van der R.C.M.; Polman, Th.H.G.; Mengelers, M.J.B.; Kuiper, H.A.

    1999-01-01

    In this study information was obtained on bioavailability of genistein, daidzein and their glycosides in human intestinal epithelial Caco-2 cells grown on semi-permeable filters. The integrity of Caco-2 monolayers was confirmed by transepithelial electrical resistance measurements and by

  2. Enterotoxigenic Escherichia coli infection and intestinal thiamin uptake: studies with intestinal epithelial Caco-2 monolayers.

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    Ghosal, Abhisek; Chatterjee, Nabendu S; Chou, Tristan; Said, Hamid M

    2013-12-01

    Infections with enteric pathogens like enterotoxigenic Escherichia coli (ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe, and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and -secreted ETEC component and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of human thiamin transporter-1 and -2 (hTHTR1 and hTHTR2) at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E. coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin and is associated with a decrease in the expression of intestinal thiamin transporters.

  3. Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

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    Milovic, Vladan; Turchanowa, Lyudmila; Stein, Jürgen; Caspary, Wolfgang F.

    2001-01-01

    AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations ( > 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco- 2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION: Transepithelial transport of putrescine across Caco-2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF. PMID:11819759

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

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    Takuya Akiyama

    Full Text Available 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.

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

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    Akiyama, Takuya; Oishi, Kenji; Wullaert, Andy

    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.

  6. Bifidobacterium lactis 420 and fish oil enhance intestinal epithelial integrity in Caco-2 cells.

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    Mokkala, Kati; Laitinen, Kirsi; Röytiö, Henna

    2016-03-01

    Increased intestinal permeability is a predisposing factor for low-grade inflammation-associated conditions, including obesity and type 2 diabetes. Dietary components may influence intestinal barrier integrity. We hypothesized that the dietary supplements Bifidobacterium lactis 420, Lactobacillus rhamnosus HN001, and fish oil have beneficial impacts on intestinal barrier integrity. In addition, we hypothesized that the coadministration of these components results in synergistic benefits to the integrity of the intestinal barrier. To study this, we investigated the impact of cell-free culture supernatant from dietary supplements B lactis 420 and L rhamnosus HN001, and fish oil, separately and in combination, on intestinal permeability in a CaCo-2 cell model. Administered separately, both B lactis 420 supernatant and fish oil significantly increased the integrity of the intestinal epithelial barrier, as determined by an increase in transepithelial electrical resistance (TEER), whereas L rhamnosus did not. The TEER increase with B lactis 420 was dose dependent. Interestingly, a combination of B lactis 420 supernatant and fish oil negated the increase in TEER of the single components. mRNA expression of tight junction proteins, measured by real-time quantitative polymerase chain reaction, was not altered, but the mRNA expression of myosin light chain kinase increased after fish oil treatment. To conclude, single dietary components, namely, B lactis 420 and fish oil, induced beneficial effects on intestinal barrier integrity in vitro, whereas a combination of 2 beneficial test compounds resulted in a null effect. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers.

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    Judit Váradi

    Full Text Available Alpha-melanocyte-stimulating hormone (α-MSH is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of α-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of α-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-α and interleukin-1β. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-κB was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. α-MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-κB p65 subunit into Caco-2 cell nuclei, which was inhibited by α-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of α-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and α-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-α and interleukin-1β cytokines.

  8. Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers.

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    Váradi, Judit; Harazin, András; Fenyvesi, Ferenc; Réti-Nagy, Katalin; Gogolák, Péter; Vámosi, György; Bácskay, Ildikó; Fehér, Pálma; Ujhelyi, Zoltán; Vasvári, Gábor; Róka, Eszter; Haines, David; Deli, Mária A; Vecsernyés, Miklós

    2017-01-01

    Alpha-melanocyte-stimulating hormone (α-MSH) is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of α-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of α-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-α and interleukin-1β. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER) and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-κB) was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. α-MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-κB p65 subunit into Caco-2 cell nuclei, which was inhibited by α-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of α-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and α-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-α and interleukin-1β cytokines.

  9. Absorption of 3(2H)-furanones by human intestinal epithelial Caco-2 cells.

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    Stadler, Nicole Christina; Somoza, Veronika; Schwab, Wilfried

    2009-05-13

    A number of 3(2H)-furanones are synthesized by fruits and have been found in cooked foodstuffs, where they impart flavor and odor because of their low perception thresholds. They show genotoxic properties in model studies but are also ranked among the antioxidants and anticarcinogens. This study examined the efficiency of intestinal absorption and metabolic conversion of 3(2H)-furanones by using Caco-2 cell monolayers as an intestinal epithelial cell model. The permeability of each agent was measured in both the apical to basal and basal to apical directions. 2,5-Dimethyl-4-methoxy-3(2H)-furanone (DMMF) showed the highest absorption rate in all experiments, while similar amounts of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), and 4-hydroxy-5-methyl-3(2H)-furanone (HMF) were taken up. HDMF-glucoside was almost not absorbed but was hydrolyzed to a small extent. The transport of 3(2H)-furanones could not be saturated even at levels of 500 microM and occurred in both directions. Because the uptake was only slightly reduced by apical hyperosmolarity, passive diffusion by paracellular transport is proposed.

  10. Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D.

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    Anabazhagan, Arivarasu N; Chatterjee, Ishita; Priyamvada, Shubha; Kumar, Anoop; Tyagi, Sangeeta; Saksena, Seema; Alrefai, Waddah A; Dudeja, Pradeep K; Gill, Ravinder K

    2017-03-16

    The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin

  11. Regulation of Intestinal Epithelial Calcium Transport Proteins by Stanniocalcin-1 in Caco2 Cells

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    Jinmei Xiang

    2016-07-01

    Full Text Available Stanniocalcin-1 (STC1 is a calcium and phosphate regulatory hormone. However, the exact molecular mechanisms underlying how STC1 affects Ca2+ uptake remain unclear. Here, the expression levels of the calcium transport proteins involved in transcellular transport in Caco2 cells were examined following over-expression or inhibition of STC1. These proteins include the transient receptor potential vanilloid members (TRPV 5 and 6, the plasma membrane calcium ATPase 1b (PMCA1b, the sodium/calcium exchanger (NCX1, and the vitamin D receptor (VDR. Both gene and protein expressions of TRPV5 and TRPV6 were attenuated in response to over-expression of STC1, and the opposite trend was observed in cells treated with siRNASTC1. To further investigate the ability of STC1 to influence TRPV6 expression, cells were treated with 100 ng/mL of recombinant human STC1 (rhSTC1 for 4 h following pre-transfection with siRNASTC1 for 48 h. Intriguingly, the increase in the expression of TRPV6 resulting from siRNASTC1 was reversed by rhSTC1. No significant effect of STC1 on the expression of PMCA1b, NCX1 or VDR was observed in this study. In conclusion, the effect of STC1 on calcium transport in intestinal epithelia is due to, at least in part, its negative regulation of the epithelial channels TRPV5/6 that mediate calcium influx.

  12. Influence of Physicochemical Properties and PEG Modification of Magnetic Liposomes on Their Interaction with Intestinal Epithelial Caco-2 Cells.

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    Kono, Yusuke; Jinzai, Hitomi; Kotera, Yota; Fujita, Takuya

    2017-12-01

    The present study aimed to investigate the effect of particle size (100, 500 nm), surface charge (cationic, neutral and anionic) and polyethylene glycol (PEG) modification of magnetic liposomes on their interaction with the human intestinal epithelial cell line, Caco-2. The cellular associated amount of all the magnetic liposomes was significantly increased by the presence of a magnetic field. The highest association and internalization into Caco-2 cells was observed with magnetic cationic liposomes. Moreover, small magnetic liposomes were more efficiently associated and taken up into the cells, than large ones. In contrast, PEG modification significantly attenuated the enhancing effect of the magnetic field on the cellular association of magnetic liposomes. We also found that magnetic cationic liposomes had the highest retention properties to Caco-2 cells. Moreover, the retention of large magnetic liposomes to the cells was much longer than that of small ones. In addition, magnetic cationic and neutral liposomes had relatively high stability in Caco-2 cells, whereas magnetic anionic liposomes rapidly degraded. These results indicate that the physicochemical properties and PEG modification of magnetic liposomes greatly influences their intestinal epithelial transport.

  13. Staphylococcus aureus induces IL-8 expression through its lipoproteins in the human intestinal epithelial cell, Caco-2.

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    Kang, Seok-Seong; Noh, Su Young; Park, Ok-Jin; Yun, Cheol-Heui; Han, Seung Hyun

    2015-09-01

    Staphylococcus aureus can cause the intestinal inflammatory diseases. However, little is known about the molecular mechanism of S. aureus infection in the intestine. In the present study, we investigated whether S. aureus could stimulate human intestinal epithelial cells triggering inflammation. When the human intestinal epithelial cell-line, Caco-2, and the primary colon cells were stimulated with ethanol-inactivated S. aureus, IL-8 expression was induced in a dose-dependent manner. The inactivated S. aureus preferentially stimulated Toll-like receptor (TLR) 2 rather than TLR4. Lipoproteins, lipoteichoic acid (LTA), and peptidoglycan (PGN) are considered as potential TLR2 ligands of S. aureus. Interestingly, S aureus lipoproteins and Pam2CSK4 mimicking Gram-positive bacterial lipoproteins, but not LTA and PGN of S. aureus, significantly induced IL-8 expression in Caco-2 cells. Furthermore, lipoprotein-deficient S. aureus mutant strain failed to induce IL-8 production. Collectively, these results suggest that S. aureus stimulates the human intestinal epithelial cells to induce the chemokine IL-8 production through its lipoproteins, potentially contributing the development of intestinal inflammation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Suppressive effect of nobiletin and epicatechin gallate on fructose uptake in human intestinal epithelial Caco-2 cells.

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    Satsu, Hideo; Awara, Sohei; Unno, Tomonori; Shimizu, Makoto

    2017-12-01

    Inhibition of excessive fructose intake in the small intestine could alleviate fructose-induced diseases such as hypertension and non-alcoholic fatty liver disease. We examined the effect of phytochemicals on fructose uptake using human intestinal epithelial-like Caco-2 cells which express the fructose transporter, GLUT5. Among 35 phytochemicals tested, five, including nobiletin and epicatechin gallate (ECg), markedly inhibited fructose uptake. Nobiletin and ECg also inhibited the uptake of glucose but not of L-leucine or Gly-Sar, suggesting an inhibitory effect specific to monosaccharide transporters. Kinetic analysis further suggested that this reduction in fructose uptake was associated with a decrease in the apparent number of cell-surface GLUT5 molecules, and not with a change in the affinity of GLUT5 for fructose. Lastly, nobiletin and ECg suppressed the permeation of fructose across Caco-2 cell monolayers. These findings suggest that nobiletin and ECg are good candidates for preventing diseases caused by excessive fructose intake.

  15. Surfactants enhance the tight-junction permeability of food allergens in human intestinal epithelial Caco-2 cells.

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    Mine, Yoshinori; Zhang, Jie Wie

    2003-02-01

    Food additives are responsible for certain allergic types of symptoms. Here Caco-2 cell monolayers were used as a model of the intestinal epithelium for the study of the effect of a food grade surfactant. We determined whether or not the presence of a surfactant enhances the transportation of food allergens across human intestinal epithelial Caco-2 cells. This study investigated sucrose monoester fatty acids, which are a food grade surfactant. As an in vitro model of human epithelial cells, Caco-2 cells were grown in monolayers and exposed to different doses of the surfactant in conjunction with ovomucoid, a major egg white allergen. The integrity of the monolayer was assessed by measuring transepithelial electrical resistance (TEER). The permeability of tight junctions and transport of the antigen were studied. TEER correlated with the permeability of tight junctions. TEER significantly decreased upon exposure to a surfactant, indicating an increase in ovomucoid permeability without degradation. The surfactant induced shortening in microvilli, actin disbandment and structural separation of tight junctions. The results indicate that food grade surfactants can increase the paracellular uptake of food allergens. Copyright 2003 S. Karger AG, Basel

  16. Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis.

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    Fenyvesi, Ferenc; Réti-Nagy, Katalin; Bacsó, Zsolt; Gutay-Tóth, Zsuzsanna; Malanga, Milo; Fenyvesi, Éva; Szente, Lajos; Váradi, Judit; Ujhelyi, Zoltán; Fehér, Pálma; Szabó, Gábor; Vecsernyés, Miklós; Bácskay, Ildikó

    2014-01-01

    Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB) on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8) cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

  17. Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis.

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    Ferenc Fenyvesi

    Full Text Available Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8 cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

  18. Genome-wide analysis of CDX2 binding in intestinal epithelial cells (Caco-2)

    DEFF Research Database (Denmark)

    Boyd, Mette; Hansen, Morten; Jensen, Tine G K

    2010-01-01

    The CDX2 transcription factor is known to play a crucial role in inhibiting proliferation, promoting differentiation and the expression of intestinal specific genes in intestinal cells. The overall effect of CDX2 in intestinal cells has previously been investigated in conditional knock-out mice, ...

  19. Anti-inflammatory properties of an extract of M. ilicifolia in the human intestinal epithelial Caco-2 cell line.

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    Wonfor, Ruth; Natoli, Manuela; Parveen, Ifat; Beckman, Manfred; Nash, Robert; Nash, Deborah

    2017-09-14

    Maytenus ilicifolia is a Celastracea plant used in traditional medicine to alleviate digestive tract inflammatory disorders. We investigated anti-inflammatory properties of M. ilicifolia crude extract towards Caco-2 cell line, as a model of Toll-like Receptor 2 (TLR-2) inflammatory pathway. Toxicity was assessed following culture of Caco-2 with M. ilicifolia, using apparent cell permeability and trans-epithelial electric resistance. Anti-inflammatory properties of M. ilicifolia were assessed through IL-8 secretion and TLR-2 associated gene expression of Caco-2 cells with or without an LTA challenge. M. ilicifolia was not toxic to Caco-2 cells. M. ilicifolia down-regulated TLR2 expression with and without LTA challenge but had no effect on other genes. Following LTA challenge of Caco-2 cells, 100 and 200µg/mL M. ilicifolia abrogated IL-8 secretion. We provide preliminary data for some M. ilicifolia anti-inflammatory properties. Further research must establish the full extent and mode of action on particular inflammatory pathways. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  20. Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers

    NARCIS (Netherlands)

    Ye, Dong; Bramini, Mattia; Hristov, Delyan R.; Wan, Sha; Salvati, Anna; Åberg, Christoffer; Dawson, Kenneth A.

    2017-01-01

    Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In

  1. The Effect of Inflammatory Status on Butyrate and Folate Uptake by Tumoral (Caco-2) and Non-Tumoral (IEC-6) Intestinal Epithelial Cells.

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    Couto, Mafalda R; Gonçalves, Pedro; Catarino, Telmo A; Martel, Fátima

    2017-01-01

    Colorectal cancer (CRC) is the second leading cause of cancer death in occidental countries. Chronic inflammatory bowel disease (crohn's disease and ulcerative colitis) is associated with an increased risk for CRC development. The aim of this work was to investigate the relationship between inflammatory status and absorption of nutrients with a role in CRC pathogenesis. In this experimental study, we evaluated the in vitro effect of tumour necrosis factor-alpha (TNF-α), interferon-γ (IF-γ), and acetylsalicylic acid on 14C-butyrate (14C- BT), 3H-folic acid (3H-FA) uptake, and on proliferation, viability and differentiation of Caco-2 and IEC-6 cells in culture. The proinflammatory cytokines TNF-α and INF-γ were found to decrease uptake of a low concentration of 14C-BT (10 µM) by Caco-2 (tumoral) and IEC-6 (normal) intestinal epithelial cell lines. However, the effect of TNF-α and INF-γ in IEC-6 cells is most probably related to a cytotoxic and antiproliferative impact. In contrast, INF-γ increases uptake of a high concentration (10 mM) of 14C-BT in Caco-2 cells. The anticarcinogenic effect of BT (10 mM) in these cells is not affected by the presence of this cytokine. On the other hand, acetylsalicylic acid stimulates 14C-BT uptake by Caco-2 cells and potentiates its antiproliferative effect. Finally, both TNF-α and INF-γ cause a significant decrease in 3H-FA uptake by Caco-2 cells. The inflammatory status has an impact upon cellular uptake of BT and FA, two nutrients with a role in CRC pathogenesis. Moreover, the anti-inflammatory acetylsalicylic acid potentiates the anticarcinogenic effect of BT in Caco-2 cells by increasing its cellular uptake.

  2. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

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    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A; Wertheimer, Joshua; Mullin, James M

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

  3. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients

    Science.gov (United States)

    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A.; Wertheimer, Joshua; Mullin, James M.

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed. PMID:26226276

  4. Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

    2017-01-01

    the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF......-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction...

  5. The pathogenic potential of Helicobacter cinaedi isolated from non-human sources: adherence, invasion and translocation ability in polarized intestinal epithelial Caco-2 cells in vitro.

    Science.gov (United States)

    Taniguchi, Takako; Yamazaki, Wataru; Saeki, Yuji; Takajo, Ichiro; Okayama, Akihiko; Hayashi, Tetsuya; Misawa, Naoaki

    2016-05-03

    Helicobacter cinaedi infection has been recognized as an increasingly important emerging disease in humans. Infection with H. cinaedi causes bacteremia, cellulitis and enteritis. H. cinaedi has been isolated from non-human sources, including dogs, cats and rodents; however, it remains unclear whether animal strains are pathogenic in humans and as zoonotic pathogens. In this study, H. cinaedi isolates were recovered from a dog and a hamster, and the ability of these isolates to adhere to, invade and translocate across polarized human intestinal epithelial Caco-2 cells was examined in vitro. To better understand the pathogenic potential of animal H. cinaedi isolates, these results were compared with those for a human strain that was isolated from a patient with bacteremia. The animal and human strains adhered to and invaded Caco-2 cells, but to a lesser degree than the C. jejuni 81-176 strain, which was used as a control. The integrity of tight junctions was monitored by measuring transepithelial electrical resistance (TER) with a membrane insert system. The TER values for all H. cinaedi strains did not change during the experimental periods compared with those of the controls; however, translocation of H. cinaedi from the apical side to the basolateral side was confirmed by cultivation and H. cinaedi-specific PCR, suggesting that the H. cinaedi strains translocated by transcellular route. This study demonstrated that H. cinaedi strains of animal origin might have a pathogenic potential in human epithelial cells as observed in a translocation assay in vitro with a human isolate.

  6. Redox-dependent modulation of lipid synthesis induced by oleic acid in the human intestinal epithelial cell line Caco-2.

    Science.gov (United States)

    Rivabene, R; Napolitano, M; Cantafora, A; Bravo, E

    2001-03-01

    The absorption, remodeling, and delivery of dietary lipids by intestinal cells are part of a complex multi-step process, the dynamics of which is influenced by the lipid composition of the diet and the physiological state of enterocytes. Emerging data indicate that, among the parameters known to modulate the cell functionality, the internal oxidative balance plays a pivotal role. In this study, we analyzed the effects of varying redox equilibria on the way in which the intestinal Caco-2 cell line utilize an exogenous lipid source such as oleic acid. Firstly, we manipulated the intracellular levels of soluble thiols (glutathione), and the amount of cell-associated products of lipid peroxidation, commonly regarded as two critical parameters characterizing the redox profile of the cells. Two different perturbants having opposite effects on the cell's redox profile were used: the pro-oxidizing agent CuSO4 (2.5 and 10 microM) and the antioxidant and thiol supplier N-acetylcysteine (NAC, 2.5 and 5 mM). The influence of these mild but critical manipulations on the incorporation of oleate (50 and 500 microM) into cholesterol, triacylglycerol, and phospholipid was then evaluated. We found that the emerging pro-oxidant condition induced by CuSO4 pre-exposure was associated with a significant up-regulation of phospholipid synthesis, while minor modifications were detected in that of triacylglycerols. Conversely, when a more reducing state was induced by NAC pre-treatment, there was a significant down-regulation of triacylglycerol synthesis, with minor modifications in that of phospholipids. In addition, the incorporation of oleic acid in the cholesteryl ester fraction appeared to be unmodified under all the redox conditions reported. On the whole, these results indicate that the pre-existing internal redox potential of the enterocytes is a critical factor that is able to differentially modulate lipid synthesis at the intestinal level. Thus, the adoption of a strategy designed

  7. Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2

    DEFF Research Database (Denmark)

    Boyd, Mette; Coskun, Mehmet; Lilje, Berit

    2014-01-01

    The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated...... genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52...... promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers....

  8. Almond milk fermented with different potentially probiotic bacteria improves iron uptake by intestinal epithelial (Caco-2) cells

    OpenAIRE

    Neus Bernat; Maite Cháfer; Amparo Chiralt; Jose Moisés Laparra; Chelo González-Martínez

    2015-01-01

    Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal [EN] New fermented almond milks were developed by using different potentially probiotic bacteria in order to cover the current demand for health-versatile non-dairy products. An in vitro digestion/Caco-2 c...

  9. Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells; assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration.

    Science.gov (United States)

    Ude, Victor C; Brown, David M; Viale, Luca; Kanase, Nilesh; Stone, Vicki; Johnston, Helinor J

    2017-08-23

    Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact. Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 μg/cm2 Cu (equivalent to 1.95 to 250 μg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 μg/cm2 for CuO NMs, and 4.25 μg/cm2 for copper sulphate (CuSO4), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO4 on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO4 on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO4 was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (Papp); a measure of barrier permeability to CuO NMs. For all experiments, CuSO4 was used as an ionic control. CuO NMs and CuSO4 caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO4 translocated across

  10. Almond milk fermented with different potentially probiotic bacteria improves iron uptake by intestinal epithelial (Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Neus Bernat

    2015-04-01

    Full Text Available New fermented almond milks were developed, using different potentially probiotic bacteria, in order to meet the current demand for healthy, versatile non-dairy products. An in vitro digestion/Caco-2 cell model was used to evaluate the effect of both non-fermented and fermented almond milks on the mitochondrial enzymatic activities of enterocytes. Moreover, macrophages were challenged with the in-vitro digested samples and the production of pro-inflammatory biomarkers TNF-a and IL-6 was quantified. Enzymatic activities of cell cultures seemed to be stimulated by the exposure to both fermented and non-fermented almond milks. Both biomarkers decreased (p< 0.05 in fermented almond milks with either B. bifidum or B. longum. Results showed that fermented almond products favored the energetic metabolism of enterocytes and had a lower inflammatory response than non-fermented almond milk, suggesting its benefits for the management of allergies/intolerances. Moreover, the fermentation process enhanced the uptake of iron by Caco-2 cells, especially when using L. rhamnosus and either B. bifidum or B. longum as starters, thus improving the product bioactivity. Therefore, new non-dairy fermented products with functional properties were developed, which might be positioned as alternatives to cow-milk products for sensitized groups of population (allergic and/or intolerant to cow milk or anemic population, among others.

  11. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

    Science.gov (United States)

    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  12. Transport of a Novel Angiotensin-I-Converting Enzyme Inhibitory Peptide Ala-His-Leu-Leu Across Human Intestinal Epithelial Caco-2 Cells.

    Science.gov (United States)

    Li, Ying; Zhao, Jiangtao; Liu, Xiaoli; Xia, Xiudong; Wang, Ying; Zhou, Jianzhong

    2017-03-01

    The transport behavior and absorption mechanism of Ala-His-Leu-Leu (AHLL) intestinal absorption in Caco-2 cell monolayers were clarified systemically. The safe absorptive concentration of AHLL was 200 μg/mL, which was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The permeation of AHLL was concentration dependent in a bidirectional transfer and reached a plateau at 90 min. The efflux ratio was above 0.5, suggesting that AHLL was absorbed by both active transport and passive diffusion. The apparent permeability coefficients (Papp) of AHLL both from the apical (AP) to basolateral (BL) side (PappAB) and from the BL to AP side (PappBA) decreased when the temperature was lowered from 37°C to 4°C.The uptake of AHLL was more at pH 7.4 than at other pHs. Both verapamil and (E)-3-[[[3-[2-(7-chloro-2- quinolinyl) ethenyl] phenyl]-[[(3-dimethyl amino)-3-oxopropyl]thio] methyl] thio]-propanoic acid (MK571) inhibited the absorption of AHLL, indicating that P-glycoprotein and multi-drug resistant proteins (MRPs) were all involved in AHLL secretion, especially multi-drug resistant protein 2 (MRP2). AHLL was transported through both trans- and paracellular pathways across the Caco-2 cell monolayer. This work first elucidates the AHLL absorption mechanism in Caco-2 cells and provides the basis for future studies on the improvement of bioavailability.

  13. EPS-SJ Exopolisaccharide Produced by the Strain Lactobacillus paracasei subsp. paracasei BGSJ2-8 Is Involved in Adhesion to Epithelial Intestinal Cells and Decrease on E. coli Association to Caco-2 Cells.

    Science.gov (United States)

    Živković, Milica; Miljković, Marija S; Ruas-Madiedo, Patricia; Markelić, Milica B; Veljović, Katarina; Tolinački, Maja; Soković, Svetlana; Korać, Aleksandra; Golić, Nataša

    2016-01-01

    The aim of this study was to determine the role of an exopolysaccharide produced by natural dairy isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8, in the adhesion to intestinal epithelial cells and a decrease in Escherichia coli's association with Caco-2 cells. Annotation of the BGSJ2-8 genome showed the presence of a gene cluster, epsSJ, which encodes the biosynthesis of the strain-specific exopolysaccharide EPS-SJ, detected as two fractions (P1 and P2) by size exclusion chromatography (SEC) coupled with multi-angle laser light scattering (MALLS) detection. SEC-MALLS analysis revealed that an EPS-SJ(-) mutant (EPS7, obtained by insertion mutagenesis of the glps_2198 gene encoding primary glycosyltransferase) does not produce the P2 fraction of EPS-SJ. Transmission electron microscopy showed that EPS7 mutant has a thinner cell wall compared to the EPS-SJ(+) strain BGSJ2-83 (a plasmid free-derivative of BGSJ2-8). Interestingly, strain BGSJ2-83 showed higher adhesion to Caco-2 epithelial intestinal cell line than the EPS7 mutant. Accordingly, BGSJ2-83 effectively reduced E. coli ATCC25922's association with Caco-2 cells, while EPS7 did not show statistically significant differences. In addition, the effect of EPS-SJ on the proliferation of lymphocytes in gastrointestinal associated lymphoid tissue (GALT) was tested and the results showed that the reduction of GALT lymphocyte proliferation was higher by BGSJ2-83 than by the mutant. To the best of our knowledge this is the first report indicating that the presence of EPS (EPS-SJ) on the surface of lactobacilli can improve communication between bacteria and intestinal epithelium, implying its possible role in gut colonization.

  14. Interleukin-10 Enhances the Intestinal Epithelial Barrier in the Presence of Corticosteroids through p38 MAPK Activity in Caco-2 Monolayers: A Possible Mechanism for Steroid Responsiveness in Ulcerative Colitis.

    Directory of Open Access Journals (Sweden)

    Violeta Lorén

    Full Text Available Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn's disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis.

  15. Germination of Bacillus cereus spores is induced by germinants from differentiated caco-2 cells, a human cell line mimicking the epithelial cells of the small intestine

    NARCIS (Netherlands)

    Wijnands, L.M.; Dufrenne, J.B.; Leusden, van F.M.; Abee, T.

    2007-01-01

    Spores of 11 enterotoxigenic strains of Bacillus cereus isolated from foods and humans adhered with similar efficiencies to Caco-2 cells, whereas subsequent germination triggering was observed with only 8 of these strains. Notably, Hep-2 cells did not trigger germination, while spores of all strains

  16. Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines.

    Science.gov (United States)

    Kouzel, Ivan U; Pohlentz, Gottfried; Schmitz, Julia S; Steil, Daniel; Humpf, Hans-Ulrich; Karch, Helge; Müthing, Johannes

    2017-10-25

    Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding precise structures of their Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), and lipid raft association. In this study we identified Gb3Cer and Gb4Cer lipoforms of serum-free cultivated Caco-2 and HCT-8 cells, chiefly harboring ceramide moieties composed of sphingosine (d18:1) and C16:0, C22:0 or C24:0/C24:1 fatty acid. The most significant difference between the two cell lines was the prevalence of Gb3Cer with C16 fatty acid in HCT-8 and Gb4Cer with C22-C24 fatty acids in Caco-2 cells. Lipid compositional analysis of detergent-resistant membranes (DRMs), which were used as lipid raft-equivalents, indicated slightly higher relative content of Stx receptor Gb3Cer in DRMs of HCT-8 cells when compared to Caco-2 cells. Cytotoxicity assays revealed substantial sensitivity towards Stx2a for both cell lines, evidencing little higher susceptibility of Caco-2 cells versus HCT-8 cells. Collectively, Caco-2 and HCT-8 cells express a plethora of different receptor lipoforms and are susceptible towards Stx2a exhibiting somewhat lower sensitivity when compared to Vero cells.

  17. Intestinal transport of Cylindrospermopsin using the Caco-2 cell line.

    Science.gov (United States)

    Pichardo, Silvia; Devesa, Vicenta; Puerto, María; Vélez, Dinoraz; Cameán, Ana M

    2017-02-01

    Cylindrospermopsin (CYN) is a cyanotoxin produced by various cyanobacterial species. It is a water soluble zwitterion, stable at extreme temperatures and pH. Despite the main route of exposure to CYN is through drinking water and food, there is a lack of data concerning its intestinal absorption and the mechanisms implicated. The aim of this study was to characterize the mechanisms involved in the intestinal absorption of CYN, using Caco-2 human cell line as a model of the intestinal epithelium. The results obtained in the present work increases the limited knowledge regarding CYN transport across the intestinal epithelium and identifies the paracellular route as an important pathway in CYN absorption. A minor carrier-mediated transcellular transport has been evidenced. This transport is not affected by low temperatures, suggesting that an active mechanism is not involved. Moreover, the transport through the intestinal monolayer is H(+) and GSH dependent and Na+independent. The transport characteristics elucidated in this study prepare the ground for future studies directed at identifying transporters involved in the intestinal absorption of this toxin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Adherence and viability of intestinal bacteria to differentiated Caco-2 cells quantified by flow cytometry.

    Science.gov (United States)

    Grootaert, Charlotte; Boon, Nico; Zeka, Fjoralba; Vanhoecke, Barbara; Bracke, Marc; Verstraete, Willy; Van de Wiele, Tom

    2011-07-01

    Recent developments in host-microbe research give rise to a growing demand for rapid and accurate methods to quantify bacterial adhesion to epithelial cells. Here, we describe a new flow cytometric method to determine the amount and viability of gut bacteria, adhered to a monolayer of differentiated cells. The latter is a more relevant epithelium model than the suspended eukaryotic cells currently used in flow cytometric protocols. During the development of the method, we monitored the adhesion potential of six bacterial species and an intestinal microbial community to Caco-2 cells. The combination of SYBR Green I/propidium iodide was more efficient than carboxyfluorescein diacetate to stain the bacterial cells. In addition, a better separation between the Caco-2 background signal and viable and dead bacteria was obtained. A precise amount of Triton X-100 was used to detach adhered bacteria from Caco-2 cells and cell debris. Yet, a limited decrease in viability was observed for the intestinal microbial community treated with Triton X-100. The flow cytometric lower detection limit for pure bacterial cultures was 3.0-4.0log/mL, whereas a 5.0-5.5log/mL detection limit was obtained in the presence of Caco-2 cell background. The latter was sufficient to quantify adhered bacteria. To the best of our knowledge, this is the first description of a flow cytometric protocol that quantifies adhesion of both pure and mixed gut microbial cultures to a differentiated monolayer of Caco-2 cells and that allows to distinguish between viable and dead adhered bacteria. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Anthocyanin Absorption and Metabolism by Human Intestinal Caco-2 Cells--A Review.

    Science.gov (United States)

    Kamiloglu, Senem; Capanoglu, Esra; Grootaert, Charlotte; Van Camp, John

    2015-09-08

    Anthocyanins from different plant sources have been shown to possess health beneficial effects against a number of chronic diseases. To obtain any influence in a specific tissue or organ, these bioactive compounds must be bioavailable, i.e., effectively absorbed from the gut into the circulation and transferred to the appropriate location within the body while still maintaining their bioactivity. One of the key factors affecting the bioavailability of anthocyanins is their transport through the gut epithelium. The Caco-2 cell line, a human intestinal epithelial cell model derived from a colon carcinoma, has been proven to be a good alternative to animal studies for predicting intestinal absorption of anthocyanins. Studies investigating anthocyanin absorption by Caco-2 cells report very low absorption of these compounds. However, the bioavailability of anthocyanins may be underestimated since the metabolites formed in the course of digestion could be responsible for the health benefits associated with anthocyanins. In this review, we critically discuss recent findings reported on the anthocyanin absorption and metabolism by human intestinal Caco-2 cells.

  20. Arctigenin from Fructus Arctii (Seed of Burdock Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers

    Directory of Open Access Journals (Sweden)

    Hee Soon Shin

    2015-01-01

    Full Text Available Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER value (as an index of barrier function and ovalbumin (OVA permeation (as an index of permeability to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function.

  1. Arctigenin from Fructus Arctii (Seed of Burdock) Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers

    Science.gov (United States)

    Shin, Hee Soon; Jung, Sun Young; Back, Su Yeon; Do, Jeong-Ryong; Shon, Dong-Hwa

    2015-01-01

    Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER) value (as an index of barrier function) and ovalbumin (OVA) permeation (as an index of permeability) to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function. PMID:26550018

  2. Evaluation of physicochemical properties and intestinal permeability of six dietary polyphenols in human intestinal colon adenocarcinoma Caco-2 cells.

    Science.gov (United States)

    Rastogi, Himanshu; Jana, Snehasis

    2016-02-01

    Phenolic compounds are common ingredients in many dietary supplements and functional foods. However, data concerning physicochemical properties and permeability of polyphenols on the intestinal epithelial cells are scarce. The aims of this study were to determine the experimental partition coefficient (Log P), and parallel artificial membrane permeability assay (PAMPA), to characterize the bi-directional transport of six phenolic compounds viz. caffeic acid, chrysin, gallic acid, quercetin, resveratrol and rutin in Caco-2 cells. The experimental Log P values of six polyphenols were correlated (R (2) = 0.92) well with the calculated Log P values. The apparent permeability (P app) range of all polyphenols in PAMPA for the apical (AP) to basolateral (BL) was 1.18 ± 0.05 × 10(-6) to 5.90 ± 0.16 × 10(-6) cm/s. The apparent Caco-2 permeability (P app) range for the AP-BL was 0.96 ± 0.03 × 10(-6) to 3.80 ± 0.45 × 10(-6) cm/s. The efflux ratio of P app (BL → AP) to P app (AP → BL) for all phenolics was Caco-2 cell monolayer permeation data. Dietary six polyphenols were poorly absorbed through PAMPA and Caco-2 cells, and their transepithelial transports were mainly by passive diffusion.

  3. The transport mechanisms of polymer nanoparticles in Caco-2 epithelial cells.

    Science.gov (United States)

    He, Bing; Lin, Ping; Jia, Zengrong; Du, Wenwen; Qu, Wei; Yuan, Lan; Dai, Wenbing; Zhang, Hua; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Zhang, Qiang

    2013-08-01

    As the primary physiological barrier, intestinal epithelial cells regulate the transportation of oral therapeutic agents including nanomedicines which significantly improves the bioavailability of many drugs. However, currently there seems in the lack of comprehensive understanding on nanoparticle transport in intestinal epithelial cells as well as the mechanisms related. So, in an attempt to illustrate the profile of nanoparticle transport in intestinal epithelial cells, Caco-2 cells and polymer nanoparticles (PNs) were used as the models to explore the whole transport process including endocytosis, intracellular trafficking, exocytosis and transcytosis. Via various techniques, the transport pathways of PNs in Caco-2 cells and their mechanisms were clarified. Firstly, the transport was characterized by its non-specificity. The co-mediation of clathrin, lipid raft/caveolae and macropinocytosis as well as the co-involvement of different proteins like actins, protein tyrosine kinase (PTK) and cyclooxygenase (COX) were found in the endocytosis of PNs. The endocytosed PNs could transport to apical early endosome (AEE) and then from AEE to lysososmes via AEE/late endosome (LE)/lysosome pathway, as well as to recycling endosome compartment (REC) or endoplasmic reticulum (ER) through AEE/REC and AEE/ER pathways, respectively. Both ER/Golgi and Golgi/REC/plasma membrane (PM) pathways were involved in the exocytosis of PNs. The transcytosis of PNs across the cell monolayer was very low with a ratio less than 0.5%, due to complicated reasons. Secondly, the transport was evidenced by its partial energy-dependency. Beside the energy-dependent transport mediated by some proteins, quantitative study demonstrated the obvious internalization as well as surface binding of PNs at both 37 °C and 4 °C, but significantly higher at 37 °C. Interestingly, the consistency between surface binding and internalization at each temperature was found, suggesting that cell binding was the

  4. Menaquinone-4 (vitamin K2) up-regulates expression of human intestinal alkaline phosphatase in Caco-2 cells.

    Science.gov (United States)

    Noda, Seiko; Yamada, Asako; Tanabe, Rieko; Nakaoka, Kanae; Hosoi, Takayuki; Goseki-Sone, Masae

    2016-11-01

    Alkaline phosphatase (ALP) hydrolyzes several monophosphate esters into inorganic acid and alcohol. In humans, 4 kinds of ALP isozymes have been identified: tissue-nonspecific ALP, intestinal ALP, placental ALP, and germ cell ALP. Intestinal ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be affected by several kinds of nutrients, such as lipids, but the physiological function of intestinal ALP has remained elusive. Vitamin K is an essential cofactor for the posttranslational carboxylation of glutamate residues into γ-carboxy glutamate. Menaquinone-4 (MK-4) with 4 isoprene units, vitamin K2, has been shown to induce bone-type ALP activity and osteoblastogenesis in human bone marrow cells. In this study, we investigated the effects of MK-4 on the level of ALP activity and expression of ALP messenger RNA in the human colon carcinoma cell line Caco-2, which is known to differentiate into small intestinal epithelial cells in vitro. After treatment with MK-4, there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments demonstrated that the increased ALP had properties of intestinal-type ALP. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that expressions of human intestinal ALP and sucrase-isomaltase, which are intestinal differentiation markers, were highly enhanced in Caco-2 cells by MK-4. This is the first report concerning ALP messenger RNA expression induced by vitamin K2 in Caco-2 cells. Further studies on the physiological functions of human intestinal ALP will provide useful data on the novel effects of vitamin K. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Efficient Adenovirus Gene Transfer Methods in Human Colonic Caco-2 Epithelial Cells Using Capric Acid.

    Science.gov (United States)

    Koizumi, Naoya; Yamagishi, Yoshiaki; Hirai, Takamasa; Fujii, Makiko; Mizuguchi, Hiroyuki; Watanabe, Yoshiteru

    2015-01-01

    Adenovirus (Ad) vectors are widely used in gene therapy and in vitro/in vivo gene transfer. However, Ad-mediated gene transfer in epithelial cells shows low efficiency, because Ad fiber cannot bind to the primary receptor, the coxsackievirus and adenovirus receptor (CAR), present in tight junctions. Caco-2 monolayer cells cultured on Transwell-chamber plates for approximately 2 weeks are widely used for drug membrane permeation studies, but Ad-mediated gene transfer is difficult in Caco-2 monolayer cells. First, we examined the efficiency of gene transfer into Caco-2 monolayer cells. Luciferase production in cultured Caco-2 cells transduced with Ad vectors was 20-fold lower on day 12 than on day 1. In contrast, the expression of CAR protein in Caco-2 cells gradually increased along with the duration of culture. For efficient gene transfer into Caco-2 monolayer cells, the binding ability of Ad vectors with CAR was found to be important. Capric acid (C10), a medium-chain fatty acid is a tight-junction modulator used as a pharmaceutical agent. We found that a novel gene transfer method using transduction with Ad vectors in the presence of C10 led more efficiently to LacZ expression in Caco-2 monolayer cells than Ad vectors alone. The results of the present study indicate that C10 could be very useful for Ad-mediated gene transfer in human colonic Caco-2 epithelial cells.

  6. Metabolism and transfer of the mycotoxin zearalenone in human intestinal Caco-2 cells.

    Science.gov (United States)

    Videmann, Bernadette; Mazallon, Michelle; Tep, Jonathan; Lecoeur, Sylvaine

    2008-10-01

    The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10-200 microM ZEA showed efficacious metabolism of the toxin. alpha-zearalenol and beta-zearalenol were the measured preponderant metabolites (respectively 40.7+/-3.1% and 31.9+/-4.9% of total metabolites, after a 3h exposure to 10 microM ZEA), whereas ZEA-glucuronide and alpha-zearalenol glucuronide were less produced (respectively 8.2+/-0.9% and 19.1+/-1.3% of total metabolites, after a 3h exposure to 10 microM ZEA). Cell production of reduced metabolites was strongly inhibited by alpha-and beta-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited alpha-hydroxysteroid dehydrogenase type II and beta-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, alpha-zearalenol was preponderantly found at the basal side, whereas beta-zearalenol and both glucuronides were preferentially excreted at the apical side. As alpha-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.

  7. Transport of Aflatoxin M1 in human intestinal Caco-2/TC7 cells

    Directory of Open Access Journals (Sweden)

    Francesca eCaloni

    2012-06-01

    Full Text Available Aflatoxin M1 (AFM1 is a hydroxylated metabolite of aflatoxin B1 (AFB1. After it is formed, it is secreted in the milk of mammals.Despite the potential risk of human exposure to AFM1, data reported in literature on the metabolism, toxicity and bioavailability of this molecule are limited and out of date. The aim of the present research was to study the absorption profile of AFM1 and possible damage to tight junctions of the intestinal Caco-2/TC7 clone grown on microporous filter supports. These inserts allowed for the separation of the apical and basolateral compartments which correspond to the in vivo lumen and the interstitial space/vascular systems of intestinal mucosa respectively.In this study, the Caco-2/TC7 cells were treated with different AFM1 concentrations (10-10,000 ng/kg for short (40 minutes and long periods of time (48 hours. The AFM1 influx/efflux transport and effects on tight junctions were evaluated by measuring trans-epithelial electrical resistance and observing tight junction protein (Zonula occludens-1 and occludin localization.The results showed that: i when introduced to the apical and basolateral compartments, AFM1 was poorly absorbed by the Caco-2/TC7 cells but its transport across the cell monolayer occurred very quickly (Papp value of 105.10 ± 7.98 cm/s x 10-6. ii The integrity of tight junctions was not permanently compromised after exposure to the mycotoxin. Viability impairment or barrier damage did not occur either.The present results contribute to the evaluation of human risk exposure to AFM1, although the AFM1 transport mechanism need to be clarified.

  8. In vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles to human epithelial colorectal adenocarcinoma (Caco-2) cells

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yijuan [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Guan, Rongfa, E-mail: rongfaguan@163.com [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Lyu, Fei [Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014 (China); Kang, Tianshu; Wu, Yihang [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Chen, Xiaoqiang [Hubei University of Technology, Wuhan 430068 (China)

    2014-11-15

    Highlights: • The characterization of Ag NPs and ZnO NPs. • The various morphologies of Caco-2 cells stained with AO/EB. • The viability of Caco-2 cells after Ag NPs and ZnO NPs exposure. • The cytotoxicity of Ag NPs and ZnO NPs on Caco-2 cells by oxidative stress assays. - Abstract: With the increasing applications of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) in foods and cosmetics, the concerns about the potential toxicities to human have been raised. The aims of this study are to observe the cytotoxicity of Ag NPs and ZnO NPs to human epithelial colorectal adenocarcinoma (Caco-2) cells in vitro, and to discover the toxicity mechanism of nanoparticles on Caco-2 cells. Caco-2 cells were exposed to 10, 25, 50, 100, 200 μg/mL of Ag NPs and ZnO NPs (90 nm). AO/EB double staining was used to characterize the morphology of the treated cells. The cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. Reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) assay were used to explore the oxidative damage of Caco-2 cells. The results showed that Ag NPs and ZnO NPs (0–200 μg/mL) had highly significant effect on the Caco-2 cells activity. ZnO NPs exerted higher cytotoxicity than Ag NPs in the same concentration range. ZnO NPs have dose-depended toxicity. The LD{sub 50} of ZnO NPs in Caco-2 cells is 0.431 mg/L. Significant depletion of SOD level, variation in GSH level and release of ROS in cells treated by ZnO NPs were observed, which suggests that cytotoxicity of ZnO NPs in intestine cells might be mediated through cellular oxidative stress. While Caco-2 cells treated with Ag NPs at all experimental concentrations showed no cellular oxidative damage. Moreover, the cells’ antioxidant capacity increased, and reached the highest level when the concentration of Ag NPs was 50 μg/mL. Therefore, it can be concluded that Ag NPs are safer antibacterial material in food packaging materials

  9. Transport of Antihypertensive Peptide RVPSL, Ovotransferrin 328-332, in Human Intestinal Caco-2 Cell Monolayers.

    Science.gov (United States)

    Ding, Long; Wang, Liying; Zhang, Yan; Liu, Jingbo

    2015-09-23

    The objective of this study was to investigate the transepithelial transport of RVPSL (Arg-Val-Pro-Ser-Leu), an egg-white-derived peptide with angiotensin I-converting enzyme (ACE) inhibitory and antihypertensive activity, in human intestinal Caco-2 cell monolayers. Results revealed that RVPSL could be passively transported across Caco-2 cell monolayers. However, during the process of transport, 36.31% ± 1.22% of the initial RVPSL added to the apical side was degraded, but this degradation decreased to 23.49% ± 0.68% when the Caco-2 cell monolayers were preincubated with diprotin A (P Caco-2 cell monolayers was studied by mutation of RVPSL. It was found that N-terminal Pro residues were more beneficial for transport of pentapeptides across Caco-2 cell monolayers than Arg and Val. Furthermore, RVPSL could be more easily transported as smaller peptides, especially in the form of dipeptides and tripeptides.

  10. Extracellular Signals of a Human Epithelial Colorectal Adenocarcinoma (Caco-2) Cell Line Facilitate the Penetration of Pseudomonas aeruginosa PAO1 Strain through the Mucin Layer.

    Science.gov (United States)

    Hayashi, Naoki; Yokotani, Atsushi; Yamamoto, Masami; Kososhi, Mariko; Morita, Mayu; Fukunishi, Chiaki; Nishizawa, Nagisa; Gotoh, Naomasa

    2017-01-01

    Pseudomonas aeruginosa can penetrate the layer of mucus formed by host intestinal epithelial cells, often resulting in sepsis in immunocompromised patients. We have previously demonstrated that P. aeruginosa can penetrate the mucin layer by flagellar motility and the degradation of the mucin layer. However, it remains unclear how P. aeruginosa initially recognizes epithelial cells. Using the human epithelial colorectal adenocarcinoma (Caco-2) cell line, we investigated extracellular signaling that could facilitate the penetration of P. aeruginosa through the mucin layer. The supernatant from Caco-2 cell cultures increased penetration of P. aeruginosa through an artificial mucin layer. The Caco-2 cell supernatant increased bacterial flagella-dependent swarming motility, but it did not influence P. aeruginosa growth or protease activity. Filtering of the Caco-2 cell supernatant indicated that proteins weighing Caco-2 cell supernatant attracted P. aeruginosa cells. Finally, we identified that growth-regulated oncogene-α (GRO-α) secreted by Caco-2 cells was a factor facilitating flagellar filament rotation and swarming motility, although it did not attract the bacteria. We conclude that penetration of the mucin layer by P. aeruginosa is facilitated by small proteins (Caco-2 cells, both by inducing acceleration of flagellar motility and increasing chemotaxis.

  11. Intestinal permeability and P-glycoprotein-mediated efflux transport of ticagrelor in Caco-2 monolayer cells.

    Science.gov (United States)

    Marsousi, Niloufar; Doffey-Lazeyras, Fabienne; Rudaz, Serge; Desmeules, Jules A; Daali, Youssef

    2016-12-01

    Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential P-glycoprotein (P-gp)-mediated active transport were assessed. To this end, bidirectional transport of ticagrelor was performed across Caco-2 (human epithelial colorectal adenocarcinoma) monolayer model in the presence and absence of potent P-gp inhibitor valspodar. Ticagrelor presented an apical-basolateral apparent permeability coefficient (Papp ) of 6.0 × 10(-6) cm/s. On the other hand, mean efflux ratio (ER) of 2.71 was observed for ticagrelor describing a higher efflux permeability compared to the influx component. Valspodar showed a significant inhibitory effect on the efflux of ticagrelor suggesting involvement of P-gp in its oral disposition. Co-incubation of the P-gp inhibitor decreased the efflux Papp of ticagrelor from 1.60 × 10(-5) to 1.13 × 10(-5) cm/s and decreased its ER by 70%. Results suggest a modest active transport of ticagrelor by P-gp across the Caco-2 cell monolayer. The co-administration of ticagrelor with a P-gp inhibitor seems altogether unlikely to have an extended impact on pharmacokinetics of ticagrelor and cause bleeding events in patients. © 2016 Société Française de Pharmacologie et de Thérapeutique.

  12. Tick-Borne Encephalitis Virus Replication, Intracellular Trafficking, and Pathogenicity in Human Intestinal Caco-2 Cell Monolayers

    Science.gov (United States)

    Möller, Lars; Schulzke, Joerg D.; Niedrig, Matthias; Bücker, Roland

    2014-01-01

    Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route. PMID

  13. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

    Directory of Open Access Journals (Sweden)

    Chao Yu

    Full Text Available Tick-borne encephalitis virus (TBEV is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin and microtubules (Nocodazole are important for PI3K-dependent (LY294002 virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1 as well as with sorting nexin-5 (SNX5, pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary

  14. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Yu, Chao; Achazi, Katharina; Möller, Lars; Schulzke, Joerg D; Niedrig, Matthias; Bücker, Roland

    2014-01-01

    Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.

  15. HNF1 alpha activates the aminopeptidase N promoter in intestinal (Caco-2) cells

    DEFF Research Database (Denmark)

    Olsen, Jørgen; Laustsen, Lotte; Troelsen, J

    1994-01-01

    The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates...... a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation....

  16. A simple coculture system shows mutualism between anaerobic faecalibacteria and epithelial Caco-2 cells.

    Science.gov (United States)

    Sadaghian Sadabad, Mehdi; von Martels, Julius Z H; Khan, Muhammed Tanweer; Blokzijl, Tjasso; Paglia, Giuseppe; Dijkstra, Gerard; Harmsen, Hermie J M; Faber, Klaas Nico

    2015-12-15

    Most gut bacteria are obligate anaerobes and are important for human health. However, little mechanistic insight is available on the health benefits of specific anaerobic gut bacteria. A main obstacle in generating such knowledge is the lack of simple and robust coculturing methods for anaerobic bacteria and oxygen-requiring human cells. Here, we describe the development of a coculture system for intestinal Caco-2 cells and an anaerobic symbiont, Faecalibacterium prausnitzii, making use of 50 mL culture tubes. F. prausnitzii was grown in 40 mL YCFAG-agar with glass-adhered Caco-2 cells placed on top in 10 mL DMEM medium. Grown for 18-36 h in a humidified incubator at 37 °C and 5% CO2, coverslip-attached Caco-2 cells promoted growth and metabolism of F. prausnitzii, while F. prausnitzii suppressed inflammation and oxidative stress in Caco-2 cells. F. prausnitzii did not compromise Caco-2 cell viability. Exogenously added porcine mucin also promoted growth of F. prausnitzii, suggesting that it may be part of the mechanism of Caco-2-stimulated growth of F. prausnitzii. This 'Human oxygen-Bacteria anaerobic' (HoxBan) coculturing system uniquely establishes host-microbe mutualism of a beneficial anaerobic gut microbe in vitro and principally allows the analysis of host-microbe interactions of pure and mixed cultures of bacteria and human cells.

  17. Usefulness of Caco-2/HT29-MTX and Caco-2/HT29-MTX/Raji B Coculture Models To Predict Intestinal and Colonic Permeability Compared to Caco-2 Monoculture.

    Science.gov (United States)

    Lozoya-Agullo, Isabel; Araújo, Francisca; González-Álvarez, Isabel; Merino-Sanjuán, Matilde; González-Álvarez, Marta; Bermejo, Marival; Sarmento, Bruno

    2017-04-03

    The Caco-2 cellular monolayer is a widely accepted in vitro model to predict human permeability but suffering from several and critical limitations. Therefore, some alternative cell cultures to mimic the human intestinal epithelium, as closely as possible, have been developed to achieve more physiological conditions, as the Caco-2/HT29-MTX coculture and the triple Caco-2/HT29-MTX/Raji B models. In this work the permeability of 12 model drugs of different Biopharmaceutical Classification System (BCS) characteristics, in the coculture and triple coculture models was assessed. Additionally, the utility of both models to classify compounds according to the BCS criteria was scrutinized. The obtained results suggested that the coculture of Caco-2/HT29-MTX and the triple coculture of Caco-2/HT29-MTX/Raji B were useful models to predict intestinal permeability and to classify the drugs in high or low permeability according to BCS. Moreover, to study thoroughly the transport mechanism of a specific drug, using a more complex model than Caco-2 monocultures is more suitable because coculture and triple coculture are more physiological models, so the results obtained with them will be closer to those obtained in the human intestine.

  18. Transport characteristics of ceftibuten, a new cephaloporin antibiotic, via the apical H+/dipeptide cotransport system in human intestinal cell line Caco-2: regulation by cell growth.

    Science.gov (United States)

    Matsumoto, S; Saito, H; Inui, K

    1995-10-01

    The intestinal epithelial cell line Caco-2 possesses the H+/dipeptide cotransport system responsible for uptake of oral cephalosporins. In this study, the transport characteristics of ceftibuten were examined from the viewpoint of cell growth in the Caco-2 cells. The uptake of cephalosporins by Caco-2 cell monolayers grown on plastic dishes was measured and analyzed kinetically. The uptake of ceftibuten was increased by lowering pH of the incubation medium and was inhibited by excess dipeptide. The transport activity of ceftibuten was dependent on the duration of analysis revealed that the development of ceftibuten uptake was due to not only a decrease in Km but also to an increase in Vmax value. The uptake of ceftibuten is mediated by the apical H+/dipeptide cotransport system which is regulated by cell growth and/or differentiation in the Caco-2 cells.

  19. In vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles to human epithelial colorectal adenocarcinoma (Caco-2) cells.

    Science.gov (United States)

    Song, Yijuan; Guan, Rongfa; Lyu, Fei; Kang, Tianshu; Wu, Yihang; Chen, Xiaoqiang

    2014-11-01

    With the increasing applications of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) in foods and cosmetics, the concerns about the potential toxicities to human have been raised. The aims of this study are to observe the cytotoxicity of Ag NPs and ZnO NPs to human epithelial colorectal adenocarcinoma (Caco-2) cells in vitro, and to discover the toxicity mechanism of nanoparticles on Caco-2 cells. Caco-2 cells were exposed to 10, 25, 50, 100, 200μg/mL of Ag NPs and ZnO NPs (90nm). AO/EB double staining was used to characterize the morphology of the treated cells. The cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. Reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) assay were used to explore the oxidative damage of Caco-2 cells. The results showed that Ag NPs and ZnO NPs (0-200μg/mL) had highly significant effect on the Caco-2 cells activity. ZnO NPs exerted higher cytotoxicity than Ag NPs in the same concentration range. ZnO NPs have dose-depended toxicity. The LD50 of ZnO NPs in Caco-2 cells is 0.431mg/L. Significant depletion of SOD level, variation in GSH level and release of ROS in cells treated by ZnO NPs were observed, which suggests that cytotoxicity of ZnO NPs in intestine cells might be mediated through cellular oxidative stress. While Caco-2 cells treated with Ag NPs at all experimental concentrations showed no cellular oxidative damage. Moreover, the cells' antioxidant capacity increased, and reached the highest level when the concentration of Ag NPs was 50μg/mL. Therefore, it can be concluded that Ag NPs are safer antibacterial material in food packaging materials than ZnO NPs. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Titanium dioxide nanoparticles activate IL8-related inflammatory pathways in human colonic epithelial Caco-2 cells

    Science.gov (United States)

    Krüger, Kristin; Cossais, François; Neve, Horst; Klempt, Martin

    2014-05-01

    Nanosized titanium dioxide (TiO2) particles are widely used as food additive or coating material in products of the food and pharmaceutical industry. Studies on various cell lines have shown that TiO2 nanoparticles (NPs) induced the inflammatory response and cytotoxicity. However, the influences of TiO2 NPs' exposure on inflammatory pathways in intestinal epithelial cells and their differentiation have not been investigated so far. This study demonstrates that TiO2 NPs with particle sizes ranging between 5 and 10 nm do not affect enterocyte differentiation but cause an activation of inflammatory pathways in the human colon adenocarcinoma cell line Caco-2. 5 and 10 nm NPs' exposures transiently induce the expression of ICAM1, CCL20, COX2 and IL8, as determined by quantitative PCR, whereas larger particles (490 nm) do not. Further, using nuclear factor (NF)-κB reporter gene assays, we show that NP-induced IL8 mRNA expression occurs, in part, through activation of NF-κB and p38 mitogen-activated protein kinase pathways.

  1. Toxoplasma gondii Infection Promotes Epithelial Barrier Dysfunction of Caco-2 Cells

    Science.gov (United States)

    Briceño, Marisol Pallete; Nascimento, Layane Alencar Costa; Nogueira, Nathalia Pires; Barenco, Paulo Victor Czarnewski; Ferro, Eloisa Amália Vieira; Rezende-Oliveira, Karine; Goulart, Luiz Ricardo; Alves, Patrícia Terra; Barbosa, Bellisa de Freitas; Lima, Wânia Rezende; Silva, Neide Maria

    2016-01-01

    After oral infection, Toxoplasma gondii invades intestinal cells, induces breakdown of intestinal physiology and barrier functions, and causes intestinal pathology in some animal species. Although parasites’ invasion into host cells is a known phenomenon, the effects of T. gondii infection in the intestinal barrier are still not well established. To evaluate morphological and physiological modifications on the colorectal adenocarcinoma-derived Caco-2 cell line during T. gondii infection, microvilli, tight junction integrity, and transepithelial electrical resistance (TEER) were investigated under infection. It was observed that the dextran uptake (endocytosis) and distribution were smaller in infected than in noninfected Caco-2 cells. The infection leads to the partial loss of microvilli at the cell surface. Claudin-1, zonula occludens-1 (ZO-1), and occludin expressions were colocalized by immunofluorescence and presented discontinuous net patterns in infected cells. Immunoblotting analysis at 24 hr postinfection revealed decreasing expression of occludin and ZO-1 proteins, whereas claudin-1 presented similar expression level compared with noninfected cells. T. gondii decreased TEER in Caco-2 cells 24 hr after infection. Our results suggest that T. gondii infection may lead to the loss of integrity of intestinal mucosa, resulting in impaired barrier function. PMID:27370796

  2. Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

    2017-01-01

    Inflammatory bowel diseases (IBD) are chronic inflammatory diseases involving all or part of the gastrointestinal tract. The stress-activated serine-threonine protein kinase D1 (PKD1) protein has previously been implicated in intestinal immune regulation. The objective of this study was to evalua...

  3. [Biotransformation by human intestinal flora and absorption-transportation characteristic in a model of Caco-2 cell monolayer of d-corydaline and tetrahydropalmatine].

    Science.gov (United States)

    Liu, Yang-Zi; Yang, Xin-Bao; Yang, Xiu-Wei; Yao, Chun-Mei; Ran, Li; Wu, Shuai; Xu, Wei; Liu, Jian-Xun

    2013-01-01

    To study the biotransformation by human intestinal flora, and the absorption and transportation characteristic in a model of human colon adenocarcinoma cell lines (Caco-2 cell) monolayer of d-corydaline (CDL) and tetrahydropalmatine (THP). CDL or THP was incubated with crude enzymes of human intestinal flora under the anaerobic environment and 37 degrees C conditions to transform CDL or THP. Caco-2 cell monolayer was used as an intestinal epithelial cell model for determination of the permeability of CDL or THP from apical side (AP side) to basolateral side (BL side) or from BL side to AP side. Transportation parameters and permeability coefficients (P(app)) were then calculated, and P(app) values were compared with the reported values for model compounds, propranolol as a well absorbed drug and atenolol as a poor absorbed drug. The concentration of CDL or THP was measured by HPLC coupled with photodiode array detector. CDL or THP in the human intestinal flora incubation system did not happen biotransformation. In the Caco-2 cell monolayer model, the P(app) magnitudes of both CDL and THP were 1 x 10(-5) cm x s(-1) in the bi-directional transport, which were identical with propranolol. And their transports were concentration dependent between 0-180 min. Both CDL and THP may be stable in the human intestinal flora incubation system, and their absorption and transportation in the human Caco-2 cell monolayer model are mainly via passive diffusion mechanism.

  4. Astilbin from Engelhardtia chrysolepis enhances intestinal barrier functions in Caco-2 cell monolayers.

    Science.gov (United States)

    Nakahara, Tatsuo; Nishitani, Yosuke; Nishiumi, Shin; Yoshida, Masaru; Azuma, Takeshi

    2017-06-05

    Astilbin, which is one of polyphenolic compounds isolated from the leaves of Engelhardtia chrysolepis HANCE (Chinese name, huang-qui), is available as the effective component in food and cosmetics because of its anti-oxidant and anti-inflammatory effects. The tight junction (TJ) proteins, which protect the body from foreign substances, are related to adhesion between a cell and a cell. Previously, the enhancement of TJ's functions induced by aglycones of flavonoids has been demonstrated, but the effects of the glycosides such as astilbin have not been observed yet. In this study, we investigated the effects of astilbin on the TJ's functions, and human colon carcinoma Caco-2 cell monolayers were used to evaluate the effects of astilbin on transepithelial electrical resistance (TER) value and the mRNA and proteins expressions of TJ-related molecules. Astilbin increased the TER value, mRNA expression levels of claudin-1 and ZO-2, and protein expression levels of occludin and ZO-2 in Caco-2 cells. Astilbin also increased the TER value in Caco-2 cells co-stimulated with TNF-α plus IFN-γ, and moreover upregulated the protein expression of TJ-related molecules in Caco-2 cells co-treated with TNF-α plus IFN-γ. These results suggest that astilbin can enhance the expressions of TJ-related molecules, leading to upregulation of the barrier functions in the intestinal cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Loss of LSR affects epithelial barrier integrity and tumor xenograft growth of CaCo-2 cells.

    Science.gov (United States)

    Czulkies, Bernd A; Mastroianni, Justin; Lutz, Lisa; Lang, Sarah; Schwan, Carsten; Schmidt, Gudula; Lassmann, Silke; Zeiser, Robert; Aktories, Klaus; Papatheodorou, Panagiotis

    2017-06-06

    The lipolysis-stimulated lipoprotein receptor (LSR) is a lipoprotein receptor, serves as host receptor for clostridial iota-like toxins and is involved in the formation of tricellular contacts. Of particular interest is the role of LSR in progression of various cancers. Here we aimed to study the tumor growth of LSR-deficient colon carcinoma-derived cell lines HCT116 and CaCo-2 in a mouse xenograft model. Whereas knockout of LSR had no effect on tumor growth of HCT116 cells, we observed that CaCo-2 LSR knockout tumors grew to a smaller size than their wild-type counterparts. Histological analysis revealed increased apoptotic and necrotic cell death in a tumor originating from LSR-deficient CaCo-2 cells. LSR-deficient CaCo-2 cells exhibited increased cell proliferation in vitro and an altered epithelial morphology with impaired targeting of tricellulin to tricellular contacts. In addition, loss of LSR reduced the transepithelial electrical resistance of CaCo-2 cell monolayers and increased permeability for small molecules. Moreover, LSR-deficient CaCo-2 cells formed larger cysts in 3D culture than their wild-type counterparts. Our study provides evidence that LSR affects epithelial morphology and barrier formation in CaCo-2 cells and examines for the first time the effects of LSR deficiency on the tumor growth properties of colon carcinoma-derived cell lines.

  6. Intestinal Transportations of Main Chemical Compositions of Polygoni Multiflori Radix in Caco-2 Cell Model

    Science.gov (United States)

    Li, Na; Lin, Pei; Li, Yunfei; Mao, Xiaojian; Bao, Getuzhaori; Zhao, Ronghua

    2014-01-01

    Context. Polygoni Multiflori Radix (PMR) is originated from the root of Polygonum multiflorum Thunb. and used in oriental countries for centuries. However, little researches pay close attention to the absorption of its major constituents. Objective. Transepithelial transport of TSG, RL, PL, and four anthraquinones is carried out. Materials and Methods. Caco-2 cell monolayer, which represented a well-established model for the study of intestinal transport of nutrients and xenobiotics, was used in this paper. Results. The apparent permeability coefficients (P app) in the Caco-2 cell monolayers were TSG (2.372 × 10−9) < EG (2.391 × 10−9) < EN (2.483 × 10−9) < PL (4.917 × 10−9) < RN (1.707 × 10−8) < RL (1.778 × 10−8) < AE (1.952 × 10−8). Thus, RN, RL, and AE were considered partly absorbed, while other constituents were hardly absorbed. Discussion and Conclusion. Glycosides showed poor permeabilities than aglycones. In the meantime, TSG and EN gave out poor recovery rates in this assay, which indicated that TSG and EN may accumulate or metabolise in the Caco-2 cells. In silico prediction indicated that Gibbs energy (r = 0.751, p < 0.05) and heat of form (r = 0.701, p < 0.05) were strongly positively correlated with P app. PMID:24693324

  7. Intestinal Transportations of Main Chemical Compositions of Polygoni Multiflori Radix in Caco-2 Cell Model

    Directory of Open Access Journals (Sweden)

    Jie Yu

    2014-01-01

    Full Text Available Context. Polygoni Multiflori Radix (PMR is originated from the root of Polygonum multiflorum Thunb. and used in oriental countries for centuries. However, little researches pay close attention to the absorption of its major constituents. Objective. Transepithelial transport of TSG, RL, PL, and four anthraquinones is carried out. Materials and Methods. Caco-2 cell monolayer, which represented a well-established model for the study of intestinal transport of nutrients and xenobiotics, was used in this paper. Results. The apparent permeability coefficients (Papp in the Caco-2 cell monolayers were TSG (2.372 × 10−9 < EG (2.391 × 10−9 < EN (2.483 × 10−9 < PL (4.917 × 10−9 < RN (1.707 × 10−8 < RL (1.778 × 10−8 < AE (1.952 × 10−8. Thus, RN, RL, and AE were considered partly absorbed, while other constituents were hardly absorbed. Discussion and Conclusion. Glycosides showed poor permeabilities than aglycones. In the meantime, TSG and EN gave out poor recovery rates in this assay, which indicated that TSG and EN may accumulate or metabolise in the Caco-2 cells. In silico prediction indicated that Gibbs energy (r=0.751, p<0.05 and heat of form (r=0.701, p<0.05 were strongly positively correlated with Papp.

  8. Chemical form of selenium affects its uptake, transport and glutathione peroxidase activity in the human intestinal Caco-2 cell model

    Science.gov (United States)

    Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources...

  9. Digestion and absorption of an egg white ACE-inhibitory peptide in human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Ding, Long; Wang, Liying; Yu, Zhipeng; Zhang, Ting; Liu, Jingbo

    2016-01-01

    The objective of this study was to investigate the digestion and absorption of egg white-derived angiotensin I-converting enzyme (ACE)-inhibitory peptide TNGIIR in human intestinal Caco-2 cell monolayers. Results showed that the digestion of TNGIIR to simulated gastrointestinal enzymes and brush border membrane peptidases were 5.87% ± 1.92% and 17.17% ± 0.64%, respectively (p Caco-2 cell monolayers was determined to be (4.92 ± 0.40) × 10(-6) cm/s, indicating that TNGIIR can transport across Caco-2 cell monolayers in intact form. In addition, only cytochalasin D, a disruptor of tight junctions (TJs), changed TNGIIR transport rate significantly (p Caco-2 cell monolayers was paracellular pathway via TJs.

  10. The Interplay between Radioresistant Caco-2 Cells and the Immune System Increases Epithelial Layer Permeability and Alters Signaling Protein Spectrum

    Science.gov (United States)

    Morini, Jacopo; Babini, Gabriele; Barbieri, Sofia; Baiocco, Giorgio; Ottolenghi, Andrea

    2017-01-01

    Colorectal cancer is one of the most frequent type of cancer, with a higher incidence in the developed countries. Colorectal cancer is usually managed with both surgeries, chemotherapy and radiotherapy. Radiotherapy has the well-known advantage of targeting the tumor, minimizing normal tissue exposure. Nevertheless, during radiation treatment, exposure of healthy tissues is of great concern, in particular because of the effects on the intestinal barrier functions and on cells belonging to the immune system. The functional role of intestinal barrier in avoiding paracellular trafficking and controlling bacterial spread from gut it is well known and it is due to the presence of tight junction complexes. However, intestinal barrier is fundamental in participating to the interplay with immune system, especially considering the gut-associated lymphoid tissue. Until few years ago, radiotherapy was considered to bear only a depressive action on the immune system. However, it is now recognized that the release of pro-inflammatory signals and phenotypic changes in tumoral cells due to ionizing radiation could trigger the immune system against the tumor. In this work, we address how intestinal barrier functions are perturbed by X-ray doses in the range 0–10 Gy, focusing on the interplay between tumoral cells and the immune system. To this aim, we adopted a coculture model in which Caco-2 cells can be grown in presence/absence of peripheral blood mononuclear cells (PBMC). We focused our attention on changes in the proliferation, trans-epithelial electrical resistance (TEER), cytokine release, and proteins of the junctional complexes. Our results indicate a high radioresistance of Caco-2 in the investigated dose range, and an increased permeability of the tumoral cell layer due to the presence of PBMC. This is found to be correlated with activation of PBMC, inhibiting the apoptotic pathway, with the enhancement of cytokine release and with variation of tight junction

  11. Protective Effects of Bifidobacterium on Intestinal Barrier Function in LPS-Induced Enterocyte Barrier Injury of Caco-2 Monolayers and in a Rat NEC Model.

    Science.gov (United States)

    Ling, Xiang; Linglong, Peng; Weixia, Du; Hong, Wei

    2016-01-01

    Zonulin protein is a newly discovered modulator which modulates the permeability of the intestinal epithelial barrier by disassembling intercellular tight junctions (TJ). Disruption of TJ is associated with neonatal necrotizing enterocolitis (NEC). It has been shown bifidobacterium could protect the intestinal barrier function and prophylactical administration of bifidobacterium has beneficial effects in NEC patients and animals. However, it is still unknown whether the zonulin is involved in the gut barrier dysfunction of NEC, and the protective mechanisms of bifidobacterium on intestinal barrier function are also not well understood. The present study aims to investigate the effects of bifidobacterium on intestinal barrier function, zonulin regulation, and TJ integrity both in LPS-induced enterocyte barrier injury of Caco-2 monolayers and in a rat NEC model. Our results showed bifidobacterium markedly attenuated the decrease in transepithelial electrical resistance and the increase in paracellular permeability in the Caco-2 monolayers treated with LPS (P bifidobacterium significantly decreased the production of IL-6 and TNF-α (P bifidobacterium pretreatment up-regulated occludin, claudin-3 and ZO-1 expression (P bifidobacterium decreased the incidence of NEC from 88 to 47% (P bifidobacterium treated rats (P bifidobacterium attenuated the increase in intestinal permeability (P bifidobacterium may protect against intestinal barrier dysfunction both in vitro and in NEC. This protective effect is associated with inhibition of proinflammatory cytokine secretion, suppression of zonulin protein release and improvement of intestinal TJ integrity.

  12. Effects of prefermented cereals or the end products of fermentation on growth and metabolism of enterocyte-like Caco-2 cells and on intestinal health of restrictedly fed weanling pigs

    NARCIS (Netherlands)

    Bruininx, E.M.A.M.; Koninkx, J.F.J.G.; Binnendijk, G.P.; Zandstra, T.; Heetkamp, M.J.W.; Peet-Schwering, van der C.M.C.; Gerrits, W.J.J.

    2010-01-01

    To unravel the underlying mechanisms that explain the positive effects of prefermented cereals on in vivo gastrointestinal (GI) architecture and function, an in vitro experiment using a human small intestinal epithelial cell model (Caco-2) was performed. A range of dilutions (0% to 10%) of the

  13. DNA damage and oxidative stress induced at low doses by the fungicide hexachlorobenzene in human intestinal Caco-2 cells.

    Science.gov (United States)

    Chalouati, Hela; Boutet, Elisa; Metais, Benjamin; Fouche, Edwin; Ben Sâad, Mohamed Moncef; Gamet-Payrastre, Laurence

    2015-01-01

    Hexachlorobenzene (HCB), a persistent chlorinated organic chemical, could be detected in human tissues in several countries of the world. Human exposure to Persistent Organic Pollutants (POPs) occurring primarily through diet, HCB and its metabolites are therefore supposed to interact directly with intestinal mucosa. The aim of this study was to investigate the possible effects of low doses of HCB on DNA integrity, cellular viability, differentiation and oxidative status in vitro in human colonic carcinoma cell line Caco-2. Cells were exposed to increasing doses of HCB for 14 days to assess the cytotoxic, genotoxic and oxidative properties of this compound. The involvement of oxidative stress in the observed effects was evaluated by co exposure of Caco-2 cells with HCB and α-tocopherol. Exposure of Caco-2 cells to HCB resulted in a dose-dependent cytotoxicity, DNA damages and alterations of the cell layer integrity and the barrier function. Moreover, exposure of Caco-2 cells to HCB led to an enhancement of H(2)O(2) production and to an increased activity of antioxidant enzymes. In addition, Co exposure of Caco-2 cells to HCB and α-tocopherol reversed the effects observed in cells exposed to HCB alone. These results suggested that HCB effects on Caco-2 cells could be linked, at least in part, to its pro-oxidative potential.

  14. The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro.

    Science.gov (United States)

    Kasper, Jennifer Y; Hermanns, Maria Iris; Cavelius, Christian; Kraegeloh, Annette; Jung, Thomas; Danzebrink, Rolf; Unger, Ronald E; Kirkpatrick, Charles James

    The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-β) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-β, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells

  15. In Situ Perfusion Model in Rat Colon for Drug Absorption Studies: Comparison with Small Intestine and Caco-2 Cell Model.

    Science.gov (United States)

    Lozoya-Agullo, Isabel; González-Álvarez, Isabel; González-Álvarez, Marta; Merino-Sanjuán, Matilde; Bermejo, Marival

    2015-09-01

    Our aim is to develop and to validate the in situ closed loop perfusion method in rat colon and to compare with small intestine and Caco-2 cell models. Correlations with human oral fraction absorbed (Fa) and human colon fraction absorbed (Fa_colon) were developed to check the applicability of the rat colon model for controlled release (CR) drug screening. Sixteen model drugs were selected and their permeabilities assessed in rat small intestine and colon, and in Caco-2 monolayers. Correlations between colon/intestine/Caco-2 permeabilities versus human Fa and human Fa_colon have been explored to check model predictability and to apply a BCS approach in order to propose a cut off value for CR screening. Rat intestine perfusion with Doluisio's method and single-pass technique provided a similar range of permeabilities demonstrating the possibility of combining data from different laboratories. Rat colon permeability was well correlated with Caco-2 cell-4 days model reflecting a higher paracellular permeability. Rat colon permeabilities were also higher than human colon ones. In spite of the magnitude differences, a good sigmoidal relationship has been shown between rat colon permeabilities and human colon fractions absorbed, indicating that rat colon perfusion can be used for compound classification and screening of CR candidates. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  16. Carrier-mediated ¿-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

    2012-01-01

    and the anticancer prodrug d-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least...

  17. Conjugated linoleic acid enhances transepithelial calcium transport in human intestinal-like Caco-2 cells: An insight into molecular changes

    NARCIS (Netherlands)

    Murphy, E.F.; Jewell, C.; Hooiveld, G.J.E.J.; Müller, M.R.; Cashman, K.D.

    2006-01-01

    Conjugated linoleic acid (CLA) has been shown to enhance paracellular and transcellular Ca transport across human intestinal-like Caco-2 cell monolayers. The mechanisms of action, however, are still unclear. Therefore, this study investigated the molecular mechanisms underlying CLA-induced

  18. Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Stetinová, Vera; Smetanová, Libuse; Kholová, Dagmar; Svoboda, Zbynek; Kvetina, Jaroslav

    2009-09-01

    This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 micromol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm). Results showed that ambroxol transport is linear with time, pH-dependent and direction-independent, displays non-saturable (first-order) kinetics. Thus, the transport seems to be transcellular mediated by passive diffusion. Estimated high solubility and high permeability (P(app) = 45 x 10(-6) cm/s) of ambroxol rank it among well absorbed compounds and class I of BCS. It can be expected that the oral dose fraction of ambroxol absorbed in human intestine is high.

  19. Insulin stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1 in the human intestinal cell line Caco-2.

    Science.gov (United States)

    Kobayashi, Taku; Koizumi, Takahiro; Kobayashi, Masaki; Ogura, Jiro; Horiuchi, Yuichi; Kimura, Yuki; Kondo, Ayuko; Furugen, Ayako; Narumi, Katsuya; Takahashi, Natsuko; Iseki, Ken

    2017-04-01

    Organic anion transporting polypeptide 2B1 (OATP2B1) is the major uptake transporter in the intestine, and transports various clinically used therapeutic agents. Insulin acts through the insulin receptor in targeted cells, and Rab8A is one of the insulin signaling pathways. The small intestine in humans also expresses insulin receptor and Rab8A. It has been reported that insulin stimulates peptide transporter 1 (PEPT1) expression at the apical membrane and increases uptake of PEPT1 substrates in small intestine epithelial model cells (Caco-2 cells). However, the effect of insulin on OATP2B1 in the small intestine has not been fully investigated. We found that Rab8A was associated with OATP2B1-mediated estrone-3-sulfate (E3S) uptake. Insulin stimulated the uptake of E3S by Caco-2 cells and the enhancement was sustained for 120 min. The Vmax value of E3S uptake significantly increased upon insulin exposure. Caco-2 cells treated with insulin showed increased OATP2B1 expression at the cell surface. The apical-to-basal transport of E3S was also increased by insulin. The increase of E3S transport was inhibited by the cold condition (4 °C) or the OATP2B1 inhibitor, taurocholate. These results indicate that insulin acts on the small intestine to increase OATP2B1-mediated absorption. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  20. Modulation of chromatin remodelling induced by the freshwater cyanotoxin cylindrospermopsin in human intestinal caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Antoine Huguet

    Full Text Available Cylindrospermopsin (CYN is a cyanotoxin that has been recognised as an emerging potential public health risk. Although CYN toxicity has been demonstrated, the mechanisms involved have not been fully characterised. To identify some key pathways related to this toxicity, we studied the transcriptomic profile of human intestinal Caco-2 cells exposed to a sub-toxic concentration of CYN (1.6 µM for 24hrs using a non-targeted approach. CYN was shown to modulate different biological functions which were related to growth arrest (with down-regulation of cdkn1a and uhrf1 genes, and DNA recombination and repair (with up-regulation of aptx and pms2 genes. Our main results reported an increased expression of some histone-modifying enzymes (histone acetyl and methyltransferases MYST1, KAT5 and EHMT2 involved in chromatin remodelling, which is essential for initiating transcription. We also detected greater levels of acetylated histone H2A (Lys5 and dimethylated histone H3 (Lys4, two products of these enzymes. In conclusion, CYN overexpressed proteins involved in DNA damage repair and transcription, including modifications of nucleosomal histones. Our results highlighted some new cell processes induced by CYN.

  1. Transport of the Glucosamine-Derived Browning Product Fructosazine (Polyhydroxyalkylpyrazine) Across the Human Intestinal Caco-2 Cell Monolayer: Role of the Hexose Transporters.

    Science.gov (United States)

    Bhattacherjee, Abhishek; Hrynets, Yuliya; Betti, Mirko

    2017-06-14

    The transport mechanism of fructosazine, a glucosamine self-condensation product, was investigated using a Caco-2 cell model. Fructosazine transport was assessed by measuring the bidirectional permeability coefficient across Caco-2 cells. The mechanism of transport was evaluated using phlorizin, an inhibitor of sodium-dependent glucose cotransporters (SGLT) 1 and 2, phloretin and quercetin, inhibitors of glucose transporters (GLUT) 1 and 2, transcytosis inhibitor wortmannin, and gap junction disruptor cytochalasin D. The role of hexose transporters was further studied using downregulated or overexpressed cell lines. The apparent permeability (P a,b ) of fructosazine was 1.30 ± 0.02 × 10 -6 cm/s. No significant (p > 0.05) effect was observed in fructosazine transport by adding wortmannin and cytochalasin D. The presence of phlorizin, phloretin, and quercetin decreased fructosazine transport. The downregulated GLUT cells line was unable to transport fructosazine. In human intestinal epithelial Caco-2 cells, GLUT1 or GLUT2 and SGLT are mainly responsible for fructosazine transport.

  2. 1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis.

    Science.gov (United States)

    Noda, Seiko; Yamada, Asako; Nakaoka, Kanae; Goseki-Sone, Masae

    2017-10-01

    Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D3, the biologically active form of vitamin D3, there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Proteomic analysis of the interaction of Bifidobacterium longum NCC2705 with the intestine cells Caco-2 and identification of plasminogen receptors.

    Science.gov (United States)

    Wei, Xiao; Yan, Xiabei; Chen, Xuannan; Yang, Zhan; Li, Huan; Zou, Dayang; He, Xiang; Wang, Simiao; Cui, Qian; Liu, Wei; Zhurina, Daria; Wang, Xuesong; Zhao, Xiangna; Huang, Liuyu; Zeng, Ming; Ye, Qinong; Riedel, Christian U; Yuan, Jing

    2014-08-28

    To identify proteins with a potential role in the interaction of Bifidobacterium longum with intestinal epithelial cells, we profiled the protein response of B. longum NCC2705 following interaction with Caco-2 cells. Thirty-one protein spots, belonging to a total of 23 proteins, which exhibited a change in abundance of at least 3-fold were identified in B. longum NCC2705 following co-culture with Caco-2 cells, and were subsequently identified. Changes in expression were confirmed at the transcriptional level for a selection of these proteins. Enolase (Eno) and elongation factor Tu (EF-Tu) were amongst the proteins that showed the most prominent increase in abundance. Interaction of these proteins with plasminogen (Plg) was analyzed by Plg overlay assays, glutathione S-transferase (GST)-pull down, and western blot analysis. The results suggested that EF-Tu and Eno serve as surface receptors for B. longum NCC2705 binding to human plasminogen. Purified GST-EF-Tu and GST-Eno inhibited adhesion of B. longum NCC2705 to Caco-2 cells. Collectively, our data suggest that Eno and EF-Tu moonlight as adhesions, and are possibly involved in the protective role played by B. longum NCC2705 in defense against enteric pathogens. Biological significance The interaction of bifidobacteria with the human host plasminogen/plasmin system confirms the existence of a new component in the molecular cross-talk between bacteria and the host. Our study analyzed proteins EF-Tu and Eno with Plg binding activity, and they can inhibit adhesion of B. longum NCC2705 to Caco-2 cells, suggesting their role in the bacterial adherent to the enterocyte surface. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Uptake of phenolic compounds from plant foods in human intestinal Caco-2 cells.

    Science.gov (United States)

    Hithamani, Gavirangappa; Kizhakayil, Dhanya; Srinivasan, Krishnapura

    2017-12-01

    In continuation of our studies on the bioaccessibility of phenolic compounds from food grains as influenced by domestic processing, we examined the uptake of phenolics from native/sprouted finger millet (Eleucine coracana) and green gram (Vigna radiata) and native/heat-processed onion (Allium cepa) in human Caco-2 cells. Absorption of pure phenolic compounds, as well as the uptake of phenolic compounds from finger millet, green gram, and onion, was investigated in Caco-2 monolayer model. Transport of individual phenolic compounds from apical compartment to the basolateral compartment across Caco-2 monolayer was also investigated. Sprouting enhanced the uptake of syringic acid from both these grains. Open-pan boiling reduced the uptake of quercetin from the onion. Among pure phenolic compounds, syringic acid was maximally absorbed, while the flavonoid isovitexin was least absorbed. Apparent permeability coefficient P(app) of phenolic compounds from their standard solutions was 2.02 x 10-6cm/s to 8.94 x 10-6cm/s. Sprouting of grains enhanced the uptake of syringic acid by the Caco-2 cells. Open-pan boiling drastically reduced the uptake of quercetin from the onion. The permeability of phenolic acids across Caco-2 monolayer was higher than those of flavonoids.

  5. Altered global gene expression profiles in human gastrointestinal epithelial Caco2 cells exposed to nanosilver

    Directory of Open Access Journals (Sweden)

    Saura C. Sahu

    2016-01-01

    Full Text Available Extensive consumer exposure to food- and cosmetics-related consumer products containing nanosilver is of public safety concern. Therefore, there is a need for suitable in vitro models and sensitive predictive rapid screening methods to assess their toxicity. Toxicogenomic profile showing subtle changes in gene expressions following nanosilver exposure is a sensitive toxicological endpoint for this purpose. We evaluated the Caco2 cells and global gene expression profiles as tools for predictive rapid toxicity screening of nanosilver. We evaluated and compared the gene expression profiles of Caco-2 cells exposed to 20 nm and 50 nm nanosilver at a concentration 2.5 μg/ml. The global gene expression analysis of Caco2 cells exposed to 20 nm nanosilver showed that a total of 93 genes were altered at 4 h exposure, out of which 90 genes were up-regulated and 3 genes were down-regulated. The 24 h exposure of 20 nm silver altered 15 genes in Caco2 cells, out of which 14 were up-regulated and one was down-regulated. The most pronounced changes in gene expression were detected at 4 h. The greater size (50 nm nanosilver at 4 h exposure altered more genes by more different pathways than the smaller (20 nm one. Metallothioneins and heat shock proteins were highly up-regulated as a result of exposure to both the nanosilvers. The cellular pathways affected by the nanosilver exposure is likely to lead to increased toxicity. The results of our study presented here suggest that the toxicogenomic characterization of Caco2 cells is a valuable in vitro tool for assessing toxicity of nanomaterials such as nanosilver.

  6. Concord and Niagara Grape Juice and Their Phenolics Modify Intestinal Glucose Transport in a Coupled in Vitro Digestion/Caco-2 Human Intestinal Model

    Directory of Open Access Journals (Sweden)

    Sydney Moser

    2016-07-01

    Full Text Available While the potential of dietary phenolics to mitigate glycemic response has been proposed, the translation of these effects to phenolic rich foods such as 100% grape juice (GJ remains unclear. Initial in vitro screening of GJ phenolic extracts from American grape varieties (V. labrusca; Niagara and Concord suggested limited inhibitory capacity for amylase and α-glucosidase (6.2%–11.5% inhibition; p < 0.05. Separately, all GJ extracts (10–100 µM total phenolics did reduce intestinal trans-epithelial transport of deuterated glucose (d7-glu and fructose (d7-fru by Caco-2 monolayers in a dose-dependent fashion, with 60 min d7-glu/d7-fru transport reduced 10%–38% by GJ extracts compared to control. To expand on these findings by assessing the ability of 100% GJ to modify starch digestion and glucose transport from a model starch-rich meal, 100% Niagara and Concord GJ samples were combined with a starch rich model meal (1:1 and 1:2 wt:wt and glucose release and transport were assessed in a coupled in vitro digestion/Caco-2 cell model. Digestive release of glucose from the starch model meal was decreased when digested in the presence of GJs (5.9%–15% relative to sugar matched control. Furthermore, transport of d7-glu was reduced 10%–38% by digesta containing bioaccessible phenolics from Concord and Niagara GJ compared to control. These data suggest that phenolics present in 100% GJ may alter absorption of monosaccharides naturally present in 100% GJ and may potentially alter glycemic response if consumed with a starch rich meal.

  7. Concord and Niagara Grape Juice and Their Phenolics Modify Intestinal Glucose Transport in a Coupled in Vitro Digestion/Caco-2 Human Intestinal Model.

    Science.gov (United States)

    Moser, Sydney; Lim, Jongbin; Chegeni, Mohammad; Wightman, JoLynne D; Hamaker, Bruce R; Ferruzzi, Mario G

    2016-07-05

    While the potential of dietary phenolics to mitigate glycemic response has been proposed, the translation of these effects to phenolic rich foods such as 100% grape juice (GJ) remains unclear. Initial in vitro screening of GJ phenolic extracts from American grape varieties (V. labrusca; Niagara and Concord) suggested limited inhibitory capacity for amylase and α-glucosidase (6.2%-11.5% inhibition; p phenolics) did reduce intestinal trans-epithelial transport of deuterated glucose (d7-glu) and fructose (d7-fru) by Caco-2 monolayers in a dose-dependent fashion, with 60 min d7-glu/d7-fru transport reduced 10%-38% by GJ extracts compared to control. To expand on these findings by assessing the ability of 100% GJ to modify starch digestion and glucose transport from a model starch-rich meal, 100% Niagara and Concord GJ samples were combined with a starch rich model meal (1:1 and 1:2 wt:wt) and glucose release and transport were assessed in a coupled in vitro digestion/Caco-2 cell model. Digestive release of glucose from the starch model meal was decreased when digested in the presence of GJs (5.9%-15% relative to sugar matched control). Furthermore, transport of d7-glu was reduced 10%-38% by digesta containing bioaccessible phenolics from Concord and Niagara GJ compared to control. These data suggest that phenolics present in 100% GJ may alter absorption of monosaccharides naturally present in 100% GJ and may potentially alter glycemic response if consumed with a starch rich meal.

  8. Effects of prefermented cereals or the end products of fermentation on growth and metabolism of enterocyte-like Caco-2 cells and on intestinal health of restrictedly fed weanling pigs

    OpenAIRE

    Bruininx, E.M.A.M.; Koninkx, J.F.J.G.; Binnendijk, G.P.; Zandstra, T.; Heetkamp, M.J.W.; Peet-Schwering, van der, C.M.C.; Gerrits, W.J.J.

    2010-01-01

    To unravel the underlying mechanisms that explain the positive effects of prefermented cereals on in vivo gastrointestinal (GI) architecture and function, an in vitro experiment using a human small intestinal epithelial cell model (Caco-2) was performed. A range of dilutions (0% to 10%) of the supernatants of three liquid experimental diets, as well as Na-lactate were used in an in vitro experiment to assess their effect on cellular growth, metabolism, differentiation and mucosal integrity us...

  9. Cobalt chloride compromises transepithelial barrier properties of CaCo-2 BBe human gastrointestinal epithelial cell layers.

    Science.gov (United States)

    DiGuilio, K M; Valenzano, M C; Rybakovsky, E; Mullin, J M

    2018-01-05

    Elevation of the transcription factor HIF-1 is a prominent mediator of not only processes that accompany hypoxia, but also the tumor microenvironment and tissue regeneration. This study uses mediators of "chemical hypoxia" to ask the question whether HIF-1α elevation in a healthy epithelial cell layer leads to leakiness in its tight junctional seals. Transepithelial electrical resistance and transepithelial diffusion of 14C-D-mannitol and other radiolabeled probes are used as indicators of transepithelial barrier function of CaCo-2 BBe human gastrointestinal epithelial cell layers cultured on permeable supports. Western immunoblot analyses of integral tight junctional proteins (occludin and claudins) are used as further indicators of barrier function change. Cobalt, an inhibitor of the prolyl hydroxylase enzymes governing HIF-1α breakdown in the cell, induces transepithelial leakiness in CaCo-2 BBe cell layers in a time and concentration-dependent manner. This increased leakiness is accompanied by significant changes in certain specific integral tight junctional (TJ) proteins such as a decreased level of occludin and increased level of claudin-5. Similar results regarding barrier function compromise also occur with other chemical inhibitors of HIF-1α breakdown, namely ciclopiroxolamine (CPX) and dimethyloxalylglycine (DMOG). The increased leak is manifested by both decreased transepithelial electrical resistance (Rt) and increased paracellular diffusion of D-mannitol (Jm). The induced transepithelial leak shows significant size selectivity, consistent with induced effects on TJ permeability. Less-differentiated cell layers were significantly more affected than well-differentiated cell layers regarding induced transepithelial leak. A genetically modified CaCo-2 variant with reduced levels of HIF-1β, showed reduced transepithelial leak in response to cobalt exposure, further indicating that elevation of HIF-1α levels induced by agents of "chemical hypoxia" is

  10. [Transport mechanism of isorhapontigenin based on human intestinal Caco-2 cells].

    Science.gov (United States)

    Yuan, Zi-Shuo; Zhang, Ting-Ting; Jin, Bo; Li, Tong; Ma, Chen

    2017-02-01

    Isorhapontigenin (ISO) is suggested to have many different kinds of pharmacology activities, such as anti-inflammatory effect, anti-oxidation effect and anti-cancer effect. This paper mainly discussed the transport mechanism of ISO in Caco-2 cell models. The concentration of ISO was determined by UPLC method with PDA detector at 310 nm, and then the apparent permeability coefficient Papp was calculated. The cytotoxic of different concentrations of ISO was investigated on Caco-2 cells to determine the concentration of drug administration. The effects of ISO concentration, time, temperature and transporter inhibitors on the transport of ISO were investigated. The test results showed that, ISO didn't have significant cytotoxicity at 10-60 μmol•L ⁻¹ in 14 hours. The transportation of ISO on Caco-2 cells was related to the concentration to a certain extent. Papp of ISO was higher than 10×10-6 cm•s ⁻¹ and ISO was absorbed easily by Caco-2 cells. The transport volume of ISO at BL side reached maximum at 3 h and was slightly decreased at 6 h. Papp (AP-BL) and Papp(BL-AP) at 4 ℃ were lower than those at 37 ℃. Papp (AP-BL) of ISO was significantly increased after adding P-gp inhibitor verapamil and Papp (BL-AP) of ISO was significantly decreased after adding MRP-2 inhibitor (probenecid or MK-571). The results suggested that transport mode of ISO was mainly passive diffusion in Caco-2 cell models, and P-gp and MRP may be involved in the transport of ISO. Copyright© by the Chinese Pharmaceutical Association.

  11. Mechanisms of Iron Uptake from Ferric Phosphate Nanoparticles in Human Intestinal Caco-2 Cells.

    Science.gov (United States)

    Perfecto, Antonio; Elgy, Christine; Valsami-Jones, Eugenia; Sharp, Paul; Hilty, Florentine; Fairweather-Tait, Susan

    2017-04-04

    Food fortification programs to reduce iron deficiency anemia require bioavailable forms of iron that do not cause adverse organoleptic effects. Rodent studies show that nano-sized ferric phosphate (NP-FePO4) is as bioavailable as ferrous sulfate, but there is controversy over the mechanism of absorption. We undertook in vitro studies to examine this using a Caco-2 cell model and simulated gastrointestinal (GI) digestion. Supernatant iron concentrations increased inversely with pH, and iron uptake into Caco-2 cells was 2-3 fold higher when NP-FePO4 was digested at pH 1 compared to pH 2. The size and distribution of NP-FePO4 particles during GI digestion was examined using transmission electron microscopy. The d50 of the particle distribution was 413 nm. Using disc centrifugal sedimentation, a high degree of agglomeration in NP-FePO4 following simulated GI digestion was observed, with only 20% of the particles ≤1000 nm. In Caco-2 cells, divalent metal transporter-1 (DMT1) and endocytosis inhibitors demonstrated that NP-FePO4 was mainly absorbed via DMT1. Small particles may be absorbed by clathrin-mediated endocytosis and micropinocytosis. These findings should be considered when assessing the potential of iron nanoparticles for food fortification.

  12. In vitro study of soil arsenic release by human gut microbiota and its intestinal absorption by Caco-2 cells.

    Science.gov (United States)

    Yin, Naiyi; Cai, Xiaolin; Du, Huili; Zhang, Zhennan; Li, Zejiao; Chen, Xiaochen; Sun, Guoxin; Cui, Yanshan

    2017-02-01

    Arsenic (As) speciation is essential in assessing health risks from As-contaminated soil. Release of soil-bound arsenic, As transformation by human gut microbiota, and the subsequent intestinal absorption of soil As metabolites were evaluated. A colon microbial community in a dynamic human gut model and the intestinal epithelial cell line Caco-2 were cultured. Arsenic speciation analysis and absorption of different As species were undertaken. In this study, soil As release (3.7-581.2 mg kg-1) was observed in the colon. Arsenic in the colon digests was transformed more quickly than that in the soil solid phase. X-ray absorption near-edge spectroscopy (XANES) analysis showed that 44.2-97.6% of arsenite [As(III)] generated due to arsenate [As(V)] reduction was in the soil solid phase after the colon phase. We observed a high degree of cellular absorption of soil As metabolites, exhibiting that the intestinal absorption of monomethylarsonic acid and As(III) (33.6% and 30.2% resp.) was slightly higher than that of dimethylarsinic acid and As(V) (25.1% and 21.7% resp.). Our findings demonstrate that human gut microbiota can directly release soil-bound arsenic, particularly As-bearing amorphous Fe/Al-oxides. Determining As transformation and intestinal absorption simultaneously will result in an accurate risk assessment of human health with soil As exposures. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. In vitro impact of five pesticides alone or in combination on human intestinal cell line Caco-2.

    Science.gov (United States)

    Ilboudo, Sylvain; Fouche, Edwin; Rizzati, Virginie; Toé, Adama M; Gamet-Payrastre, Laurence; Guissou, Pierre I

    2014-01-01

    In Burkina Faso, as in most Sahelian countries, the failure to follow good agricultural practices coupled with poor soil and climate conditions in the locust control context lead to high environmental contaminations with pesticide residues. Thus, consumers being orally exposed to a combination of multiple pesticide residues through food and water intake, the digestive tract is a tissue susceptible to be directly exposed to these food contaminants. The aim of our work was to compare in vitro the impact of five desert locust control pesticides (Deltamethrin DTM, Fenitrothion FNT, Fipronil FPN, Lambda-cyalothrine LCT, and Teflubenzuron TBZ) alone and in combination on the human intestinal Caco-2 cells viability and function. Cells were exposed to 0.1-100 μM pesticides for 10 days alone or in mixture (MIX). Our results showed a cytotoxic effect of DTM, FNT, FPN, LCT, and TBZ alone or in combination in human intestinal Caco-2 cells. The most efficient were shown to be FPN and FNT impacting the cell layer integrity and/or barrier function, ALP activity, antioxidant enzyme activity, lipid peroxidation, Akt activation, and apoptosis. The presence of antioxidant reduced lipid peroxidation level and attenuated the pesticides-induced cell toxicity, suggesting that key mechanism of pesticides cytotoxicity may be linked to their pro-oxidative potential. A comparative analysis with the predicted cytotoxic effect of pesticides mixture using mathematical modeling shown that the combination of these pesticides led to synergistic effects rather than to a simple independent or dose addition effect.

  14. On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Nourrisson, C; Wawrzyniak, I; Cian, A; Livrelli, V; Viscogliosi, E; Delbac, F; Poirier, P

    2016-11-01

    Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.

  15. Regional Expression Levels of Drug Transporters and Metabolizing Enzymes along the Pig and Human Intestinal Tract and Comparison with Caco-2 Cells.

    Science.gov (United States)

    Vaessen, Stefan F C; van Lipzig, Marola M H; Pieters, Raymond H H; Krul, Cyrille A M; Wortelboer, Heleen M; van de Steeg, Evita

    2017-04-01

    Intestinal transporter proteins and metabolizing enzymes play a crucial role in the oral absorption of a wide variety of drugs. The aim of the current study was to characterize better available intestinal in vitro models by comparing expression levels of these proteins and enzymes between porcine intestine, human intestine, and Caco-2 cells. We therefore determined the absolute protein expression of 19 drug transporters and the mRNA expression of 12 metabolic enzymes along the pig intestinal tract (duodenum, jejunum, ileum; N = 4), in human intestine (jejunum; N = 9), and Caco-2 cells. Expression of the included transporters and enzymes was in general well comparable between porcine and human intestinal tissue, although breast cancer resistance protein, monocarboxylate transporter 5, multidrug resistance protein (MRP) 1, MRP1, MRP3 (∼2-fold), and organic anion-transporting polypeptide (OATP) 4A1 (∼6-fold) was higher expressed in pig compared with human jejunum. Alternatively, expression level of relevant transporter proteins (glucose transporter 1, OATP4A1, MRP2, MRP1, and OATP2B1) was significantly higher (3- to 130-fold) in Caco-2 cells compared with human jejunum. Moreover, all examined CYPs showed at least a fivefold lower gene expression in Caco-2 cells compared with human jejunum, with the smallest differences for CYP1A1 and CYP3A5 and the largest difference for CYP3A4 (871-fold higher expression in human jejunum compared with Caco-2 cells). In conclusion, a comprehensive overview is provided of the expression levels of clinically relevant transporter proteins and metabolic enzymes in porcine and human intestinal tissue and Caco-2 cells, which may assist in deciding upon the most suitable model to further improve our understanding of processes that determine intestinal absorption of compounds. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Effects of polyethylene glycols on intestinal efflux pump expression and activity in Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Darya Hodaei

    2015-09-01

    Full Text Available The present study was planned to investigate the influence of polyethylene glycols (PEGs on the activity and expression of P-glycoprotein (P-gp. Sub-toxic concentrations of PEGs in Caco-2 cells were determined using the MTT test assay. Then the measurement of Rhodamine-123 (Rho-123 uptake, a P-gp fluorescence substrate, in Caco-2 cells confronting PEG 400 (1% and 2% w/v, PEG 4000 (2% and 4% w/v, PEG 6000 (2% and 4% w/v, PEG 10000 (2% and 4% w/v, PEG 15000 (1% and 2% w/v, and PEG 35000 (2% and 4% w/v overnight was taken to elucidate whether non-toxic concentrations of PEGs are able to impact P-gp activity. Furthermore, western blotting was carried out to investigate P-gp protein expression. The results showed that PEG 400 at concentrations of 1% (w/v and 2% (w/v and PEG 6000 at the concentration of 4% (w/v are notably capable of blocking P-gp. Based on the obtained results it is concluded that the mentioned excipients could be used to obstruct P-gp efflux transporter in order to increase the bioavailability of co-administered substrate drug.

  17. Establishment and Characterization of a Novel Caco-2 Subclone with a Similar Low Expression Level of Human Carboxylesterase 1 to Human Small Intestine.

    Science.gov (United States)

    Ohura, Kayoko; Nishiyama, Hikaru; Saco, Saori; Kurokawa, Keisuke; Imai, Teruko

    2016-12-01

    Caco-2 cells predominantly express human carboxylesterase 1 (hCE1), unlike the human intestine that predominantly expresses human carboxylesterase 2 (hCE2). Transport experiments using Caco-2 cell monolayers often lead to misestimation of the intestinal absorption of prodrugs because of this difference, as prodrugs designed to increase the bioavailability of parent drugs are made to be resistant to hCE2 in the intestine, so that they can be hydrolyzed by hCE1 in the liver. In the present study, we tried to establish a new Caco-2 subclone, with a similar pattern of carboxylase expression to human intestine, to enable a more accurate estimation of the intestinal absorption of prodrugs. Although no subclone could be identified with high expression levels of only hCE2, two subclones, #45 and #78, with extremely low expression levels of hCE1 were subcloned from parental Caco-2 cells by the limiting dilution technique. Unfortunately, subclone #45 did not form enterocyte-like cell monolayers due to low expression of claudins and β-actin. However, subclone #78 formed polarized cell monolayers over 4 weeks and showed similar paracellular and transcellular transport properties to parental Caco-2 cell monolayers. In addition, the intestinal transport of oseltamivir, a hCE1 substrate, could be evaluated in subclone #78 cell monolayers, including P-glycoprotein-mediated efflux under nonhydrolysis conditions, unlike parental Caco-2 cells. Consequently, it is proposed that subclone #78 may provide a more effective system in which to evaluate the intestinal absorption of prodrugs that are intended to be hydrolyzed by hCE1. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Are additive effects of dietary surfactants on intestinal tight junction integrity an overlooked human health risk? - A mixture study on Caco-2 monolayers.

    Science.gov (United States)

    Glynn, Anders; Igra, Annachiara Malin; Sand, Salomon; Ilbäck, Nils Gunnar; Hellenäs, Karl Erik; Rosén, Johan; Aspenström-Fagerlund, Bitte

    2017-08-01

    Surfactants may cause dysfunction of intestinal tight junctions (TJs), which is a common feature of intestinal autoimmune diseases. Effects of dietary surfactants on TJ integrity, measured as trans-epithelial resistance (TEER), were studied in Caco-2 cell monolayers. Cytotoxicity was assessed as apical LDH leakage. Monolayers were apically exposed for 60 min to the dietary surfactants solanine and chaconine (SC, potato glycoalkaloids, 0-0.25 mM), perfluorooctane sulfonic acid (PFOS, industrial contaminant, 0-0.8 mM), and sucrose monolaurate (SML, food emulsifier E 473, 0-2.0 mM) separately and as a mixture. Dose-response modelling of TEER EC50 showed that SC were 2.7- and 12-fold more potent than PFOS and SML, respectively. The mixture was composed of 1 molar unit SC, 2.7 units PFOS and 12 units SML ("SC TEER equivalent" proportions 1:1:1). Mixture exposure (0-0.05 mM SC equivalents) dose-response modelling suggested additive action on TJ integrity. Increasing SC and SML concentrations caused increased LDH leakage, but PFOS decreased LDH leakage at intermediate exposure concentrations. In the mixture PFOS appeared to protect from extensive SC- and SML-induced LDH leakage. Complex mixtures of surfactants in food may act additively on intestinal TJ integrity, which should be considered in risk assessment of emulsifier authorisation for use in food production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. The utility of in vitro trials that use Caco-2 cell systems as a replacement for animal intestinal permeability and human bioequivalence measurements in drug development

    Directory of Open Access Journals (Sweden)

    Margarida Estudante

    2015-04-01

    Full Text Available Caco-2 cells have been widely used for in vitro intestinal permeability screening of new molecules in drug development but with some pitfalls. Limiting the application of Caco-2 permeability screening to passive compounds is difficult as the majority of approved drugs include both passive diffusion and active transport. The aim of this study was to evaluate Caco-2 cells utility in assessing effects of P-gp mediated efflux. For that purpose the study design included the highly soluble, highly permeable (class 1, verapamil and diltiazem, the highly soluble and poorly permeable drug (class 3 digoxin and the P-gp inhibitor GG918. The apparent permeability and efflux ratio (ER were calculated. Digoxin, a positive control for P-gp, presented an ER of 4, which decreased to around 1 by GG918 addition, consistent with a P-gp effect in Caco-2 cells. ER for verapamil and diltiazem was nearly 1 and the presence of GG918 resulted in no ERs changes. These results suggest that P-gp apparently plays a minimal role in transport of class 1 drugs across Caco-2 cells while class 3 drugs should be significantly affected by P-gp. It is suggested that Caco-2 cells may be useful to determine whether P-gp plays a relevant role in intestinal absorption.

  20. Apple extract induces increased epithelial resistance and claudin 4 expression in Caco-2 cells

    NARCIS (Netherlands)

    Vreeburg, R.A.M.; Wezel, van E.E.; Ocana-Calahorro, F.; Mes, J.J.

    2012-01-01

    BACKGROUND: The small intestinal epithelium functions both to absorb nutrients, and to provide a barrier between the outside, luminal, world and the human body. One of the passageways across the intestinal epithelium is paracellular diffusion, which is controlled by the properties of tight junction

  1. In vitro impact of five pesticides alone or in combination on human intestinal cell line Caco-2

    Directory of Open Access Journals (Sweden)

    Sylvain Ilboudo

    2014-01-01

    Full Text Available In Burkina Faso, as in most Sahelian countries, the failure to follow good agricultural practices coupled with poor soil and climate conditions in the locust control context lead to high environmental contaminations with pesticide residues. Thus, consumers being orally exposed to a combination of multiple pesticide residues through food and water intake, the digestive tract is a tissue susceptible to be directly exposed to these food contaminants. The aim of our work was to compare in vitro the impact of five desert locust control pesticides (Deltamethrin DTM, Fenitrothion FNT, Fipronil FPN, Lambda-cyalothrine LCT, and Teflubenzuron TBZ alone and in combination on the human intestinal Caco-2 cells viability and function. Cells were exposed to 0.1–100 μM pesticides for 10 days alone or in mixture (MIX. Our results showed a cytotoxic effect of DTM, FNT, FPN, LCT, and TBZ alone or in combination in human intestinal Caco-2 cells. The most efficient were shown to be FPN and FNT impacting the cell layer integrity and/or barrier function, ALP activity, antioxidant enzyme activity, lipid peroxidation, Akt activation, and apoptosis. The presence of antioxidant reduced lipid peroxidation level and attenuated the pesticides-induced cell toxicity, suggesting that key mechanism of pesticides cytotoxicity may be linked to their pro-oxidative potential. A comparative analysis with the predicted cytotoxic effect of pesticides mixture using mathematical modeling shown that the combination of these pesticides led to synergistic effects rather than to a simple independent or dose addition effect.

  2. Ginger Extract Suppresses Inflammatory Response and Maintains Barrier Function in Human Colonic Epithelial Caco-2 Cells Exposed to Inflammatory Mediators.

    Science.gov (United States)

    Kim, Yunyoung; Kim, Dong-Min; Kim, Ji Yeon

    2017-05-01

    The beneficial effects of ginger in the management of gastrointestinal disturbances have been reported. In this study, the anti-inflammatory potential of ginger extract was assessed in a cellular model of gut inflammation. In addition, the effects of ginger extract and its major active compounds on intestinal barrier function were evaluated. The response of Caco-2 cells following exposure to a mixture of inflammatory mediators [interleukin [IL]-1β, 25 ng/mL; lipopolysaccharides [LPS], 10 ng/mL; tumor necrosis factor [TNF]-α, 50 ng/mL; and interferon [INF]-γ, 50 ng/mL] were assessed by measuring the levels of secreted IL-6 and IL-8. In addition, the mRNA levels of cyclooxygenase-2 and inducible nitric oxide synthase were measured. Moreover, the degree of nuclear factor (NF)-κB inhibition was examined, and the intestinal barrier function was determined by measuring the transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran transfer. It was observed that ginger extract and its constituents improved inflammatory responses by decreasing the levels of nitrite, PGE2, IL-6, and IL-8 via NF-κB inhibition. The ginger extract also increased the TEER and decreased the transfer of FITC-dextran from the apical side of the epithelium to the basolateral side. Taken together, these results show that ginger extract may be developed as a functional food for the maintenance of gastrointestinal health. © 2017 Institute of Food Technologists®.

  3. Intestinal toxicity evaluation of TiO2 degraded surface-treated nanoparticles: a combined physico-chemical and toxicogenomics approach in caco-2 cells

    Directory of Open Access Journals (Sweden)

    Fisichella Matthieu

    2012-05-01

    Full Text Available Abstract Background Titanium dioxide (TiO2 nanoparticles (NPs are widely used due to their specific properties, like UV filters in sunscreen. In that particular case TiO2 NPs are surface modified to avoid photocatalytic effects. These surface-treated nanoparticles (STNPs spread in the environment and might release NPs as degradation residues. Indeed, degradation by the environment (exposure to UV, water and air contact … will occur and could profoundly alter the physicochemical properties of STNPs such as chemistry, size, shape, surface structure and dispersion that are important parameters for toxicity. Although the toxicity of surface unmodified TiO2 NPs has been documented, nothing was done about degraded TiO2 STNPs which are the most likely to be encountered in environment. The superoxide production by aged STNPs suspensions was tested and compared to surface unmodified TiO2 NPs. We investigated the possible toxicity of commercialized STNPs, degraded by environmental conditions, on human intestinal epithelial cells. STNPs sizes and shape were characterized and viability tests were performed on Caco-2 cells exposed to STNPs. The exposed cells were imaged with SEM and STNPs internalization was researched by TEM. Gene expression microarray analyses were performed to look for potential changes in cellular functions. Results The production of reactive oxygen species was detected with surface unmodified TiO2 NPs but not with STNPs or their residues. Through three different toxicity assays, the STNPs tested, which have a strong tendency to aggregate in complex media, showed no toxic effect in Caco-2 cells after exposures to STNPs up to 100 μg/mL over 4 h, 24 h and 72 h. The cell morphology remained intact, attested by SEM, and internalization of STNPs was not seen by TEM. Moreover gene expression analysis using pangenomic oligomicroarrays (4x 44000 genes did not show any change versus unexposed cells after exposure to 10 μg/ mL, which

  4. Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells

    NARCIS (Netherlands)

    Roubos-van den Hil, P.J.; Nout, M.J.R.; Beumer, R.R.; Meulen, van der J.; Zwietering, M.H.

    2009-01-01

    Aims: This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and

  5. In vitro intestinal bioavailability of arsenosugar metabolites and presystemic metabolism of thio-dimethylarsinic acid in Caco-2 cells.

    Science.gov (United States)

    Leffers, Larissa; Wehe, Christoph A; Hüwel, Sabine; Bartel, Marc; Ebert, Franziska; Taleshi, Mojtaba S; Galla, Hans-Joachim; Karst, Uwe; Francesconi, Kevin A; Schwerdtle, Tanja

    2013-08-01

    Whereas inorganic arsenic is classified as a human carcinogen, risks to human health related to the presence of arsenosugars in marine food are still unclear. Since studies indicate that human inorganic arsenic metabolites contribute to inorganic arsenic induced carcinogenicity, a risk assessment for arsenosugars should also include a toxicological characterization of their respective metabolites. Here we assessed intestinal bioavailability of the human arsenosugar metabolites oxo-DMAA(V), thio-DMAA(V), oxo-DMAE(V), thio-DMAE(V) and thio-DMA(V) in relation to arsenite in the Caco-2 intestinal barrier model. Whereas arsenite and thio-DMA(V) caused barrier disruption at concentrations ≥10 μM, all other metabolites did not cause a barrier leakage, even when applied at 50 times higher concentrations than arsenite and thio-DMA(V). The transfer studies point to a strong intestinal bioavailability of thio-DMA(V) and thio-DMAE(V), whereas oxo-DMAA(V), thio-DMAA(V) and oxo-DMAE(V) passed the in vitro intestinal barrier only to a very small extent. Detailed influx and efflux studies indicate that arsenite and thio-DMA(V) cross the intestinal barrier most likely by passive diffusion (paracellular) and facilitated (transcellular) transport. LC-ICP-QMS based arsenic speciation studies during the transfer experiments demonstrate transfer of thio-DMA(V) itself across the intestinal barrier and suggest metabolism of thio-DMA(V) using the in vitro intestinal barrier model to its oxygen-analogue DMA(V). In the case of arsenite no metabolism was observed. In summary the two arsenosugar metabolites thio-DMA(V) and thio-DMAE(V) showed intestinal bioavailability similar to that of arsenite, and about 10-fold higher than that reported for arsenosugars (Leffers et al., Mol. Nutr. Food Res., 2013, DOI: 10.1002/mnfr.201200821) in the same in vitro model. Thus, a presystemic metabolism of arsenosugars might strongly impact arsenic intestinal bioavailability after arsenosugar intake and

  6. T84 monolayers are superior to Caco-2 as a model system of colonocytes.

    Science.gov (United States)

    Devriese, Sarah; Van den Bossche, Lien; Van Welden, Sophie; Holvoet, Tom; Pinheiro, Iris; Hindryckx, Pieter; De Vos, Martine; Laukens, Debby

    2017-07-01

    Colonic adenocarcinoma-derived Caco-2 and T84 epithelial cell lines are frequently used as in vitro model systems of functional epithelial barriers. Both are utilised interchangeably despite evidence that differentiated Caco-2 cells are more reminiscent of small intestinal enterocytes than of colonocytes, whereas differentiated T84 cells are less well characterised. The aim of this study was, therefore, to further characterise and compare differentiated Caco-2 and T84 cells. The objectives were to (1) compare the brush border morphology, (2) measure the expression of enterocyte- and colonocyte-specific genes and (3) compare their response to butyrate, which is dependent on the monocarboxylate transporter 1 (MCT1), an apical protein expressed primarily in colonocytes. T84 microvilli were significantly shorter than those of Caco-2 cells, which is a characteristic difference between small intestinal enterocytes and colonocytes. Also, enterocyte-associated brush border enzymes expressed in differentiated Caco-2 cells were not increased during T84 maturation, whereas colonic markers such as MCT1 were more abundant in differentiated T84 cells compared to differentiated Caco-2 cells. Consequently, T84 cells displayed a dose-responsive improvement of barrier function towards butyrate, which was absent in Caco-2 cells. On the other hand, differences in epithelial toll-like receptor expression between Caco-2 and T84 monolayers did not result in a corresponding differential functional response. We conclude that differentiated Caco-2 and T84 cells have distinct morphological, biochemical and functional characteristics, suggesting that T84 cells do not acquire the biochemical signature of mature small intestinal enterocytes like Caco-2 cells, but retain much of their original colonic characteristics throughout differentiation. These findings can help investigators select the appropriate intestinal epithelial cell line for specific in vitro research purposes.

  7. The effect of onion exposure on gene expression profiles in intestinal Caco-2 cells

    NARCIS (Netherlands)

    Wit, de N.J.W.; Govers, C.C.F.M.; Boekschoten, M.V.; Mes, J.J.

    2016-01-01

    Background: Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, in this study we explored the applicability of an in vitro model, namely human

  8. Polyphenols and phenolic acids from strawberry and apple decrease glucose uptake and transport by human intestinal Caco-2 cells.

    Science.gov (United States)

    Manzano, Susana; Williamson, Gary

    2010-12-01

    The effect of polyphenols, phenolic acids and tannins (PPTs) from strawberry and apple on uptake and apical to basolateral transport of glucose was investigated using Caco-2 intestinal cell monolayers. Substantial inhibition on both uptake and transport was observed by extracts from both strawberry and apple. Using sodium-containing (glucose transporters SGLT1 and GLUT2 both active) and sodium-free (only GLUT2 active) conditions, we show that the inhibition of GLUT2 was greater than that of SGLT1. The extracts were analyzed and some of the constituent PPTs were also tested. Quercetin-3-O-rhamnoside (IC₅₀ =31 μM), phloridzin (IC₅₀=146 μM), and 5-caffeoylquinic acid (IC₅₀=2570 μM) contributed 26, 52 and 12%, respectively, to the inhibitory activity of the apple extract, whereas pelargonidin-3-O-glucoside (IC₅₀=802 μM) contributed 26% to the total inhibition by the strawberry extract. For the strawberry extract, the inhibition of transport was non-competitive based on kinetic analysis, whereas the inhibition of cellular uptake was a mixed-type inhibition, with changes in both V(max) and apparent K(m) . The results in this assay show that some PPTs inhibit glucose transport from the intestinal lumen into cells and also the GLUT2-facilitated exit on the basolateral side. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer.

    Science.gov (United States)

    Yu, Yingxin; Wang, Mengmeng; Zhang, Kaiqiong; Yang, Dan; Zhong, Yufang; An, Jing; Lei, Bingli; Zhang, Xinyu

    2017-04-01

    Oral ingestion plays an important role in human exposure to polybrominated diphenyl ethers (PBDEs). The uptake of PBDEs primarily occurs in the small intestine. The aim of the present study is to investigate the transepithelial transport characteristics and mechanisms of PBDEs in the small intestine using a Caco-2 cell monolayer model. The apparent permeability coefficients of PBDEs indicated that tri- to hepta-BDEs were poorly absorbed compounds. A linear increase in transepithelial transport was observed with various concentrations of PBDEs, which suggested that passive diffusion dominated their transport at the concentration range tested. In addition, the pseudo-first-order kinetics equation can be applied to the transepithelial transport of PBDEs. The rate-determining step in transepithelial transport of PBDEs was trans-cell transport including the trans-pore process. The significantly lower transepithelial transport rates at low temperature for bidirectional transepithelial transport suggested that an energy-dependent transport mechanism was involved. The efflux transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) and influx transporters (organic cation transporters) participated in the transepithelial transport of PBDEs. In addition, the transepithelial transport of PBDEs was pH sensitive; however, more information is required to understand the influence of pH. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Characterization of loxoprofen transport in Caco-2 cells: the involvement of a proton-dependent transport system in the intestinal transport of loxoprofen.

    Science.gov (United States)

    Narumi, Katsuya; Kobayashi, Masaki; Kondo, Ayuko; Furugen, Ayako; Yamada, Takehiro; Takahashi, Natsuko; Iseki, Ken

    2016-11-01

    Loxoprofen, a propionate non-steroidal anti-inflammatory drug (NSAID), is used widely in East Asian countries. However, little is known about the transport mechanisms contributing to its intestinal absorption. The objectives of this study were to characterize the intestinal transport of loxoprofen using the human intestinal Caco-2 cell model. The transport of loxoprofen was investigated in cellular uptake studies. The uptake of loxoprofen into Caco-2 cells was pH- and concentration-dependent, and was described by a Michaelis-Menten equation with passive diffusion (Km : 4.8 mm, Vmax : 142 nmol/mg protein/30 s, and Kd : 2.2 μl/mg protein/30 s). Moreover, the uptake of loxoprofen was inhibited by a typical monocarboxylate transporter (MCT) inhibitor as well as by various monocarboxylates. The uptake of [(14) C] l-lactic acid, a typical MCT substrate, in Caco-2 cells was saturable with relatively high affinity for MCT. Because loxoprofen inhibited the uptake of [(14) C] l-lactic acid in a noncompetitive manner, it was unlikely that loxoprofen uptake was mediated by high-affinity MCT(s). Our results suggest that transport of loxoprofen in Caco-2 cells is, at least in part, mediated by a proton-dependent transport system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Use of Microarray Datasets to generate Caco-2-dedicated Networks and to identify Reporter Genes of Specific Pathway Activity

    NARCIS (Netherlands)

    Venkatasubramanian, Prashanna Balaji; Toydemir, Gamze; Wit, de Nicole; Saccenti, Edoardo; Martins dos Santos, Vitor A.P.; Baarlen, van Peter; Wells, Jerry M.; Suarez-Diez, Maria; Mes, Jurriaan J.

    2017-01-01

    Intestinal epithelial cells, like Caco-2, are commonly used to study the interaction between food, other luminal factors and the host, often supported by microarray analysis to study the changes in gene expression as a result of the exposure. However, no compiled dataset for Caco-2 has ever been

  12. Study of the Biotransformation of Tongmai Formula by Human Intestinal Flora and Its Intestinal Permeability across the Caco-2 Cell Monolayer.

    Science.gov (United States)

    Wu, Shuai; Xu, Wei; Wang, Fu-Rong; Yang, Xiu-Wei

    2015-10-15

    Tongmai formula (TMF) is a well-known Chinese medicinal preparation that contains isoflavones as its major bioactive constituents. As traditional Chinese medicines (TCMs) are usually used by oral administration, their fate inside the intestinal lumen, including their biotransformation by human intestinal flora (HIF) and intestinal absorption deserves study. In this work TMF extract was incubated with human intestinal bacteria under anaerobic conditions and the changes in the twelve main constituents of TMF were then investigated. Their intestinal permeabilities, i.e., the transport capability across the intestinal brush border were investigated with a human colon carcinoma cell line (Caco-2) cell monolayer model to predict the absorption mechanism. Meanwhile, rapid HPLC-DAD methods were established for the assay. According to the biotransformation curves of the twelve constituents and the permeability coefficients, the intestinal absorption capacity of the typical compounds was elevated from the levels of 10(-7) cm/s to 10(-5) cm/s from those of the original compounds in TMF. Among them the main isoflavone glycosides puerarin (4), mirificin (6) and daidzin (7) were transformed into the same aglycone, daidzein (10). Therefore it was predicted that the aglycone compounds might be the real active ingredients in TMF. The models used can represent a novel path for the TCM studies.

  13. Adaptation in Caco-2 Human Intestinal Cell Differentiation and Phenolic Transport with Chronic Exposure to Blackberry (Rubus sp.) Extract.

    Science.gov (United States)

    Redan, Benjamin W; Albaugh, George P; Charron, Craig S; Novotny, Janet A; Ferruzzi, Mario G

    2017-04-05

    As evidence mounts for a health-protective role of dietary phenolics, the importance of understanding factors influencing bioavailability increases. Recent evidence has suggested chronic exposure to phenolics may impact their absorption and metabolism. To explore alterations occurring from chronic dietary exposure to phenolics, Caco-2 cell monolayers were differentiated on Transwell inserts with 0-10 μM blackberry (Rubus sp.) total phenolics extracts rich in anthocyanins, flavonols, and phenolic acids. Following differentiation, apical to basolateral transport of phenolics was assessed from an acute treatment of 100 μM blackberry phenolics from 0 to 4 h. Additionally, differences in gene expression of transport and phase II metabolizing systems including ABC transporters, organic anion transporters (OATs), and uridine 5'-diphospho (UDP) glucuronosyltransferases (UGTs) were probed. After 4 h, 1 μM pretreated monolayers showed a significant (P transport including less epicatechin (42.1 ± 0.53), kaempferol glucoside (23.5 ± 0.29), and dicaffeoylquinic acid (31.9 ± 0.20) compared to control. Finally, significant (P transport proteins were observed with treatment. Therefore, adaptation to blackberry extract exposure may impact intestinal transport and metabolism of phenolics.

  14. A tunable Caco-2/HT29-MTX co-culture model mimicking variable permeabilities of the human intestine obtained by an original seeding procedure.

    Science.gov (United States)

    Béduneau, Arnaud; Tempesta, Camille; Fimbel, Stéphane; Pellequer, Yann; Jannin, Vincent; Demarne, Frédéric; Lamprecht, Alf

    2014-07-01

    Standard monoculture models utilizing Caco-2 monolayers were extensively used to mimic the permeability of the human intestinal barrier. However, they exhibit numerous limitations such as the lack of mucus layer, an overestimation of the P-gp-mediated efflux and a low paracellular permeability. Here, we suggest a new procedure to set up an in vitro model of intestinal barrier to adjust gradually the properties of the absorption barrier. Mucin-secreting HT29-MTX cells were added to Caco-2 absorptive cells in a Transwell® at different time intervals. Effects of seeding day of HT29-MTX on the paracellular permeability of lucifer yellow (LY) and on the P-gp-mediated efflux of rhodamine 123 were investigated. Apparent permeability of the rhodamine 123 in the secretory direction was highly dependent on the seeding day of goblet cells. Transepithelial electrical resistance values and LY transport across the co-cultures in the apical-to-basolateral direction were intermediary between single Caco-2 and HT29-MTX models. Early seeding days of HT29-MTX allowed increasing the fraction of goblet cells in the co-culture. Co-culture permeability was unchanged between 21 and 30 days after Caco-2 seeding, corresponding to the period of use for Caco-2-based cell models. Thus, the HT29-MTX seeding day was a key factor to set up an in vitro intestinal model with tailor-made barrier properties in terms of P-gp expression and paracellular permeability. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. The Small Colony Variant Of Listeria Monocytogenes Is More Tolerant To Antibiotics And Grows Better Within Caco-2 Epithelial Cells Than The Wild Type

    DEFF Research Database (Denmark)

    Curtis, Thomas; Gram, Lone; Knudsen, Gitte Maegaard

    2015-01-01

    tolerant of 20mM H2O2 as compared to the wild type, with 6.3 log10 CFU/ml and 3.7 log10 CFU/ml, respectively. The SCV E18 had lower survival rate in unactivated macrophages, however, it was able to survive and multiply to almost 100-fold higher CFU/ml than the wild type in CaCo-2 epithelial cells...

  16. Antibodies against Lewis antigens inhibit the binding of human norovirus GII.4 virus-like particles to saliva but not to intestinal Caco-2 cells.

    Science.gov (United States)

    Carmona-Vicente, Noelia; Allen, David J; Rodríguez-Díaz, Jesús; Iturriza-Gómara, Miren; Buesa, Javier

    2016-05-21

    Human noroviruses (NoVs) are the main cause of gastroenteritis worldwide. The most commonly detected NoV strains belong to the genetically diverse GII.4 genotype, with new pandemic variants emerging periodically. Despite extensive efforts, NoV investigation has been hampered by the lack of an effective in vitro cell culture system. However, NoV-derived recombinant virus-like particles (VLPs) resembling empty capsids are good surrogates for analysing NoV antigenicity and virus-ligand interactions. NoV VLPs have been reported to bind to histo-blood group antigens (HBGAs). We have analysed the ability of NoV VLPs derived from GI.1 genotype and from three GII.4 genotype variants, GII.4-1999, GII.4-2004 and GII.4-2006b, to bind to porcine gastric mucin (PGM), human saliva and differentiated human intestinal Caco-2 cells (D-Caco-2 cells). Distinct patterns of saliva binding with the NoV GII.4 variant VLPs were observed, although they bound to D-Caco-2 cells independently of the expression of HBGAs. Monoclonal antibodies against Lewis antigens were able to block the binding of NoV VLPs to saliva, but not to D-Caco-2 cells. Blocking HBGAs on the surface of D-Caco-2 cells with specific monoclonal antibodies did not affect NoV VLP binding to cellular membranes. Co-localisation of Lewis y (Le(y)) and H-type 2 antigens with NoV VLPs was not observed by immunofluorescence assays. Although the binding of NoV VLPs of GII.4 genotype variants to human saliva samples occur with distinct HBGA binding patterns and can be blocked by antibodies against Lewis antigens, their attachment to D-Caco-2 cells can be mediated by other receptors, which still need further investigation.

  17. Short communication: Early-lactation, but not mid-lactation, bovine lactoferrin preparation increases epithelial barrier integrity of Caco-2 cell layers.

    Science.gov (United States)

    Anderson, Rachel C; Bassett, Shalome A; Haggarty, Neill W; Gopal, Pramod K; Armstrong, Kelly M; Roy, Nicole C

    2017-02-01

    Bovine lactoferrin is an important milk protein with many health-promoting properties, including improving intestinal barrier integrity. Dysfunction of this barrier, commonly referred to as "leaky gut," has been linked to inflammatory and autoimmune diseases. With some processing techniques, lactoferrin isolated from milk collected at the start of the milking season (early lactation) may have lower purity than that isolated from milk collected during the rest of the milking season (mid-lactation) and could result in differences in bioactivity based on the stage of lactation. We compared reversed-phase HPLC chromatographs of early-lactation and mid-lactation preparations and found that both had large chromatograph peaks at the time predicted for lactoferrin. The notable difference between the 2 chromatographs was a much larger peak in the early-lactation lactoferrin sample that was determined to be angiogenin. Angiogenin was first identified due to its ability to induce new blood vessel formation, but is now known to be involved in numerous physiological processes. Then, we compared the effects of early-lactation and mid-lactation lactoferrin preparations in 2 bioassays: trans-epithelial electrical resistance (TEER), a measure of intestinal barrier integrity, and peripheral blood mononuclear cell cytokine secretion, a measure of immune-stimulatory properties. We found that early-lactation lactoferrin increased TEER across Caco-2 cell layers compared with control from 10 to 48 h, mid-lactation lactoferrin did not alter TEER. We also found that early-lactation lactoferrin reduced the amount of IL-8 produced by peripheral blood mononuclear cells (compared with those treated with control medium) to a greater extent than mid-lactation lactoferrin. A pro-inflammatory chemokine, IL-8 is also known to decrease barrier function. These results suggest that the decrease in IL-8 production in the presence of early-lactation lactoferrin may be the mechanism by which it increases

  18. CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

    Directory of Open Access Journals (Sweden)

    Geneviève Mailhot

    2010-05-01

    Full Text Available Abnormal fatty acid composition (FA in plasma and tissue lipids frequently occurs in homozygous and even in heterozygous carriers of cystic fibrosis transmembrane conductance regulator (CFTR mutations. The mechanism(s underlying these abnormalities remained, however, poorly understood despite the potentially CFTR contributing role.The aim of the present study was to investigate the impact of CFTR depletion on FA uptake, composition and metabolism using the intestinal Caco-2/15 cell line. shRNA-mediated cftr gene silencing induced qualitative and quantitative modifications in FA composition in differentiated enterocytes as determined by gas-liquid chromatography. With the cftr gene disruption, there was a 1,5 fold increase in the total FA amount, largely attributable to monounsaturated and saturated FA compared to controls. The activity of delta-7 desaturase, estimated by the 16:1(n-7/16:0, was significantly higher in knockdown cells and consistent with the striking elevation of the n-7 FA family. When incubated with [14C]-oleic acid, CFTR-depleted cells were capable of quick incorporation and export to the medium concomitantly with the high protein expression of L-FABP known to promote intracellular FA trafficking. Accordingly, lipoprotein vehicles (CM, VLDL, LDL and HDL, isolated from CFTR knockdown cells, exhibited higher levels of radiolabeled FA. Moreover, in the presence of [14C]-acetate, knockdown cells exhibited enhanced secretion of newly synthesized phospholipids, triglycerides, cholesteryl esters and free FA, thereby suggesting a stimulation of the lipogenic pathway. Conformably, gene expression of SREBP-1c, a key lipogenic transcription factor, was increased while protein expression of the phosphorylated and inactive form of acetylCoA carboxylase was reduced, confirming lipogenesis induction. Finally, CFTR-depleted cells exhibited lower gene expression of transcription factors (PPARalpha, LXRalpha, LXRbeta and RXRalpha

  19. Transepithelial Transport of YWDHNNPQIR and Its Metabolic Fate with Cytoprotection against Oxidative Stress in Human Intestinal Caco-2 Cells.

    Science.gov (United States)

    Xu, Feiran; Wang, Lifeng; Ju, Xingrong; Zhang, Jing; Yin, Shi; Shi, Jiayi; He, Rong; Yuan, Qiang

    2017-03-15

    Studies on antioxidant peptides extracted from foodstuff sources have included not only experiments to elucidate their chemical characteristics but also to investigate their bioavailability and intracellular mechanisms. This study was designed to clarify the absorption and antioxidative activity of YWDHNNPQIR (named RAP), which is derived from rapeseed protein using a Caco-2 cell transwell model. Results showed that 0.8% RAP (C0 = 0.2 mM, t = 90 min) could maintain the original structure across the Caco-2 cell monolayers via the intracellular transcytosis pathway, and the apparent drug absorption rate (Papp) was (6.6 ± 1.24) × 10-7 cm/s. Three main fragments (WDHNNPQIR, DHNNPQIR, and YWDHNNPQ) and five modified peptides derived from RAP were found in both the apical and basolateral side of the Caco-2 cell transwell model. Among these new metabolites, WDHNNPQIR had the greatest antioxidative activity in Caco-2 cells apart from the DPPH assay. With a RAP concentration of 200 μM, there were significant differences in four antioxidative indicators (T-AOC, GSH-Px, SOD, and MDA) compared to the oxidative stress control (P Caco-2 cells, which was caused by AAPH-induced oxidative damage.

  20. Use of Microarray Datasets to generate Caco-2-dedicated Networks and to identify Reporter Genes of Specific Pathway Activity.

    Science.gov (United States)

    Venkatasubramanian, Prashanna Balaji; Toydemir, Gamze; de Wit, Nicole; Saccenti, Edoardo; Martins Dos Santos, Vitor A P; van Baarlen, Peter; Wells, Jerry M; Suarez-Diez, Maria; Mes, Jurriaan J

    2017-07-28

    Intestinal epithelial cells, like Caco-2, are commonly used to study the interaction between food, other luminal factors and the host, often supported by microarray analysis to study the changes in gene expression as a result of the exposure. However, no compiled dataset for Caco-2 has ever been initiated and Caco-2-dedicated gene expression networks are barely available. Here, 341 Caco-2-specific microarray samples were collected from public databases and from in-house experiments pertaining to Caco-2 cells exposed to pathogens, probiotics and several food compounds. Using these datasets, a gene functional association network specific for Caco-2 was generated containing 8937 nodes 129711 edges. Two in silico methods, a modified version of biclustering and the new Differential Expression Correlation Analysis, were developed to identify Caco-2-specific gene targets within a pathway of interest. These methods were subsequently applied to the AhR and Nrf2 signalling pathways and altered expression of the predicted target genes was validated by qPCR in Caco-2 cells exposed to coffee extracts, known to activate both AhR and Nrf2 pathways. The datasets and in silico method(s) to identify and predict responsive target genes can be used to more efficiently design experiments to study Caco-2/intestinal epithelial-relevant biological processes.

  1. Effects of Nitric Oxide and Reactive Oxygen Species on HIF-1α Stabilization Following Clostridium Difficile Toxin Exposure of the Caco-2 Epithelial Cell Line

    Directory of Open Access Journals (Sweden)

    Joshua Y. Lee

    2013-08-01

    Full Text Available Background/Aims: Stabilization of the hypoxia-inducible factor (HIF-1α is proposed to provide a protective host-response to C. difficile intoxication. Here, we aimed to elucidate whether nitric oxide and/or reactive oxygen species produced during C. difficile toxin exposure could influence HIF-1α stability and initiate protection against epithelial cell damage. Methods/Results: HIF-1α and inducible nitric oxide synthase (iNOS proteins were up-regulated whereas factor-inhibiting HIF-1 (FIH-1 protein was down-regulated in Caco-2 epithelial cell monolayers with in vitro toxin exposure. We demonstrate using the biotin-switch assay that the stabilization of HIF-1α protein occurred via iNOS-dependent nitrosylation. Inhibition of iNOS activity by selective inhibitor (1400W attenuated HIF-1α stabilization and exacerbated toxin-dependent disruptions in Caco-2 monolayer morphology and tight junctional integrity in vitro. Treatment of Caco-2 cell monolayers with N-actylcysteine (NAC, a scavenger of reactive oxygen species (ROS, attenuated toxin-dependent increases in iNOS and HIF-1α protein levels but had no effect on FIH-1 responses. In addition, mice that were exposed to C. difficile toxin in vivo also demonstrated a significant increase in HIF-1α protein and nitrosylation levels. Conclusion: Taken together, these data suggest that important synergistic actions exist between nitric oxide and ROS to stabilize HIF-1α and its innate, protective actions in the context of C. difficile toxin-mediated epithelial injury.

  2. Diamine Oxidase from White Pea (Lathyrus sativus) Combined with Catalase Protects the Human Intestinal Caco-2 Cell Line from Histamine Damage.

    Science.gov (United States)

    Jumarie, Catherine; Séïde, Marilyne; Marcocci, Lucia; Pietrangeli, Paola; Mateescu, Mircea Alexandru

    2017-07-01

    Diamine oxidase (DAO) administration has been proposed to treat certain gastrointestinal dysfunctions induced by histamine, an immunomodulator, signaling, and pro-inflammatory factor. However, H2O2 resulting from the oxidative deamination of histamine by DAO may be toxic. The purpose of this study was to investigate to which extent DAO from white pea (Lathyrus sativus), alone or in combination with catalase, may modulate histamine toxicity in the human intestinal Caco-2 cell line. The results show that histamine at concentrations higher than 1 mM is toxic to the Caco-2 cells, independently of the cell differentiation status, with a LC50 of ≅ 10 mM following a 24-h exposure. Depending on its concentration, DAO increased histamine toxicity to a greater extent in differentiated cells compared to undifferentiated cultures. In the presence of catalase, the DAO-induced increase in histamine toxicity was completely abolished in the undifferentiated cells and only partially decreased in differentiated cells, showing differences in the sensitivity of Caco-2 cells to the products resulting from histamine degradation by DAO (H2O2, NH3, or imidazole aldehyde). It appears that treatment of food histaminosis using a combination of vegetal DAO and catalase would protect against histamine toxicity and prevent H2O2-induced damage that may occur during histamine oxidative deamination.

  3. Evaluation of the intestinal permeability of rosemary (Rosmarinus officinalis L.) extract polyphenols and terpenoids in Caco-2 cell monolayers

    Science.gov (United States)

    Arráez-Román, David; González-Álvarez, Isabel; Ibáñez, Elena; Segura-Carretero, Antonio; Bermejo, Marival; Micol, Vicente

    2017-01-01

    Rosemary (Rosmarinus officinalis) is grown throughout the world and is widely used as a medicinal herb and to season and preserve food. Rosemary polyphenols and terpenoids have attracted great interest due to their potential health benefits. However, complete information regarding their absorption and bioavailability in Caco-2 cell model is scarce. The permeation properties of the bioactive compounds (flavonoids, diterpenes, triterpenes and phenylpropanoids) of a rosemary extract (RE), obtained by supercritical fluid extraction, was studied in Caco-2 cell monolayer model, both in a free form or liposomed. Compounds were identified and quantitated by liquid chromatography coupled to quadrupole time-of-flight with electrospray ionization mass spectrometry analysis (HPLC-ESI-QTOF-MS), and the apparent permeability values (Papp) were determined, for the first time in the extract, for 24 compounds in both directions across cell monolayer. For some compounds, such as triterpenoids and some flavonoids, Papp values found were reported for the first time in Caco-2 cells.Our results indicate that most compounds are scarcely absorbed, and passive diffusion is suggested to be the primary mechanism of absorption. The use of liposomes to vehiculize the extract resulted in reduced permeability for most compounds. Finally, the biopharmaceutical classification (BCS) of all the compounds was achieved according to their permeability and solubility data for bioequivalence purposes. BCS study reveal that most of the RE compounds could be classified as classes III and IV (low permeability); therefore, RE itself should also be classified into this category. PMID:28234919

  4. Evaluation of the intestinal permeability of rosemary (Rosmarinus officinalis L.) extract polyphenols and terpenoids in Caco-2 cell monolayers.

    Science.gov (United States)

    Pérez-Sánchez, Almudena; Borrás-Linares, Isabel; Barrajón-Catalán, Enrique; Arráez-Román, David; González-Álvarez, Isabel; Ibáñez, Elena; Segura-Carretero, Antonio; Bermejo, Marival; Micol, Vicente

    2017-01-01

    Rosemary (Rosmarinus officinalis) is grown throughout the world and is widely used as a medicinal herb and to season and preserve food. Rosemary polyphenols and terpenoids have attracted great interest due to their potential health benefits. However, complete information regarding their absorption and bioavailability in Caco-2 cell model is scarce. The permeation properties of the bioactive compounds (flavonoids, diterpenes, triterpenes and phenylpropanoids) of a rosemary extract (RE), obtained by supercritical fluid extraction, was studied in Caco-2 cell monolayer model, both in a free form or liposomed. Compounds were identified and quantitated by liquid chromatography coupled to quadrupole time-of-flight with electrospray ionization mass spectrometry analysis (HPLC-ESI-QTOF-MS), and the apparent permeability values (Papp) were determined, for the first time in the extract, for 24 compounds in both directions across cell monolayer. For some compounds, such as triterpenoids and some flavonoids, Papp values found were reported for the first time in Caco-2 cells.Our results indicate that most compounds are scarcely absorbed, and passive diffusion is suggested to be the primary mechanism of absorption. The use of liposomes to vehiculize the extract resulted in reduced permeability for most compounds. Finally, the biopharmaceutical classification (BCS) of all the compounds was achieved according to their permeability and solubility data for bioequivalence purposes. BCS study reveal that most of the RE compounds could be classified as classes III and IV (low permeability); therefore, RE itself should also be classified into this category.

  5. Evaluation of the intestinal permeability of rosemary (Rosmarinus officinalis L. extract polyphenols and terpenoids in Caco-2 cell monolayers.

    Directory of Open Access Journals (Sweden)

    Almudena Pérez-Sánchez

    Full Text Available Rosemary (Rosmarinus officinalis is grown throughout the world and is widely used as a medicinal herb and to season and preserve food. Rosemary polyphenols and terpenoids have attracted great interest due to their potential health benefits. However, complete information regarding their absorption and bioavailability in Caco-2 cell model is scarce. The permeation properties of the bioactive compounds (flavonoids, diterpenes, triterpenes and phenylpropanoids of a rosemary extract (RE, obtained by supercritical fluid extraction, was studied in Caco-2 cell monolayer model, both in a free form or liposomed. Compounds were identified and quantitated by liquid chromatography coupled to quadrupole time-of-flight with electrospray ionization mass spectrometry analysis (HPLC-ESI-QTOF-MS, and the apparent permeability values (Papp were determined, for the first time in the extract, for 24 compounds in both directions across cell monolayer. For some compounds, such as triterpenoids and some flavonoids, Papp values found were reported for the first time in Caco-2 cells.Our results indicate that most compounds are scarcely absorbed, and passive diffusion is suggested to be the primary mechanism of absorption. The use of liposomes to vehiculize the extract resulted in reduced permeability for most compounds. Finally, the biopharmaceutical classification (BCS of all the compounds was achieved according to their permeability and solubility data for bioequivalence purposes. BCS study reveal that most of the RE compounds could be classified as classes III and IV (low permeability; therefore, RE itself should also be classified into this category.

  6. Retrograde cholesterol transport in the human Caco-2/TC7 cell line: a model to study trans-intestinal cholesterol excretion in atherogenic and diabetic dyslipidemia.

    Science.gov (United States)

    Dugardin, Camille; Briand, Olivier; Touche, Véronique; Schonewille, Marleen; Moreau, François; Le May, Cédric; Groen, Albert K; Staels, Bart; Lestavel, Sophie

    2017-02-01

    The dyslipidemia associated with type 2 diabetes is a major risk factor for the development of atherosclerosis. Trans-intestinal cholesterol excretion (TICE) has recently been shown to contribute, together with the classical hepatobiliary route, to fecal cholesterol excretion and cholesterol homeostasis. The aim of this study was to develop an in vitro cell model to investigate enterocyte-related processes of TICE. Differentiated Caco-2/TC7 cells were grown on transwells and incubated basolaterally (blood side) with human plasma and apically (luminal side) with lipid micelles. Radioactive and fluorescent cholesterol tracers were used to investigate cholesterol uptake at the basolateral membrane, intracellular distribution and apical excretion. Our results show that cholesterol is taken up at the basolateral membrane, accumulates intracellularly as lipid droplets and undergoes a cholesterol acceptor-facilitated and progressive excretion through the apical membrane of enterocytes. The overall process is abolished at 4 °C, suggesting a biologically active phenomenon. Moreover, this trans-enterocytic retrograde cholesterol transport displays some TICE features like modulation by PCSK9 and an ABCB1 inhibitor. Finally, we highlight the involvement of microtubules in the transport of plasma cholesterol from basolateral to apical pole of enterocytes. The human Caco-2/TC7 cell line appears a good in vitro model to investigate the enterocytic molecular mechanisms of TICE, which may help to identify intestinal molecular targets to enhance reverse cholesterol transport and fight against dyslipidemia.

  7. Determination of S-methyl-L-methionine (SMM) from Brassicaceae Family Vegetables and Characterization of the Intestinal Transport of SMM by Caco-2 Cells.

    Science.gov (United States)

    Song, Ji-Hoon; Lee, Hae-Rim; Shim, Soon-Mi

    2017-01-01

    The objectives of the current study were to determine S-methyl-L-methionine (SMM) from various Brassicaceae family vegetables by using validated analytical method and to characterize the intestinal transport mechanism of SMM by the Caco-2 cells. The SMM is well known to provide therapeutic activity in peptic ulcers. The amount of SMM from various Brassicaceae family vegetables ranged from 89.08 ± 1.68 μg/g to 535.98 ± 4.85 μg/g of dry weight by using validated ultra-performance liquid chromatography-electrospray ionization-mass spectrometry method. For elucidating intestinal transport mechanism, the cells were incubated with or without transport inhibitors, energy source, or a metabolic inhibitor. Phloridzin and verapamil as inhibitors of sodium glucose transport protein (SGLT1) and P-glycoprotein, respectively, were not responsible for cellular uptake of SMM. Glucose and sodium azide were not affected by the cellular accumulation of SMM. The efflux ratio of SMM was 0.26, implying that it is not effluxed through Caco-2 cells. The apparent coefficient permeability (Papp ) of SMM was 4.69 × 10(-5) cm/s, indicating that it will show good oral absorption in in vivo. © 2016 Institute of Food Technologists®.

  8. Effects of Marine Oils, Digested with Human Fluids, on Cellular Viability and Stress Protein Expression in Human Intestinal Caco-2 Cells

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    Cecilia Tullberg

    2017-11-01

    Full Text Available In vitro digestion of marine oils has been reported to promote lipid oxidation, including the formation of reactive aldehydes (e.g., malondialdehyde (MDA and 4-hydroxy-2-hexenal (HHE. We aimed to investigate if human in vitro digestion of supplemental levels of oils from algae, cod liver, and krill, in addition to pure MDA and HHE, affect intestinal Caco-2 cell survival and oxidative stress. Cell viability was not significantly affected by the digests of marine oils or by pure MDA and HHE (0–90 μM. Cellular levels of HSP-70, a chaperone involved in the prevention of stress-induced protein unfolding was significantly decreased (14%, 28%, and 14% of control for algae, cod and krill oil, respectively; p ≤ 0.05. The oxidoreductase thioredoxin-1 (Trx-1 involved in reducing oxidative stress was also lower after incubation with the digested oils (26%, 53%, and 22% of control for algae, cod, and krill oil, respectively; p ≤ 0.001. The aldehydes MDA and HHE did not affect HSP-70 or Trx-1 at low levels (8.3 and 1.4 μM, respectively, whilst a mixture of MDA and HHE lowered Trx-1 at high levels (45 μM, indicating less exposure to oxidative stress. We conclude that human digests of the investigated marine oils and their content of MDA and HHE did not cause a stress response in human intestinal Caco-2 cells.

  9. Prosaposin knockdown in Caco-2 cells decreases cellular levels of coenzyme Q10 and ATP, and results in the loss of tight junction barriers.

    Science.gov (United States)

    Kashiba, Misato; Terashima, Masayuki; Sagawa, Tomofumi; Yoshimura, Shinichi; Yamamoto, Yorihiro

    2017-03-01

    Coenzyme Q10 (CoQ10) is a key component of the mitochondrial electron transfer chain and is one of the most important antioxidants. We previously found that glycoprotein prosaposin (Psap) binds CoQ10 in human cells. Although Psap is expressed in the intestines, its role in the gastrointestinal tract is not clear. To elucidate the role of Psap in the intestines, we established Psap knockdown (KD) Caco-2 cells, which are an intestinal epithelial cell line. Cellular CoQ10 levels decreased significantly in Psap KD Caco-2 cells as compared to parental Caco-2 cells. Cellular ATP levels also decreased significantly in Psap KD Caco-2 cells as compared to parental Caco-2 cells. Lower ATP levels in the intestines have been reported to result in the failure of tight junction formation. As expected, Psap KD Caco-2 monolayers did not produce transepithelial electrical resistance, while parental Caco-2 monolayers did. Moreover, a fluorescent dye, lucifer yellow, leaked out through Psap KD Caco-2 monolayers, whereas it did not through parental Caco-2 monolayers. These results indicate that Psap is essential to maintain cellular levels of CoQ10 and ATP, and consequently to form tight junctions in the gastrointestinal tract.

  10. Anti-inflammatory effects of pomegranate (Punica granatum L.) husk ellagitannins in Caco-2 cells, an in vitro model of human intestine.

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    Hollebeeck, Sylvie; Winand, Julie; Hérent, Marie-France; During, Alexandrine; Leclercq, Joëlle; Larondelle, Yvan; Schneider, Yves-Jacques

    2012-08-01

    This study aimed at evaluating the anti-inflammatory properties of a pomegranate fruit husk (PomH) polyphenolic extract, rich in punicalagin, using Caco-2 cells, an in vitro model of human intestinal epithelium. Differentiated cells in bicameral inserts were pretreated or not with a PomH extract or punicalagin, as reference, at the apical side, representing the intestinal lumen. Inflammation was then induced with a cocktail of cytokines (Il-1β, TNFα and IFNγ) and LPS. After 24 h incubation, 3 pro-inflammatory markers, i.e., interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1, were assayed both at their gene transcription (qRT-PCR) and secretion (ELISA) levels. As previously described, the pro-inflammatory cocktail significantly stimulated these 3 markers, at the gene transcript and secretion levels. In inflamed cells, a significant down-regulation of the transcription of the genes encoding IL-6 and MCP-1 was observed in the presence of the PomH extract or punicalagin, while IL-8 transcription was unaffected. Both treatments also decreased the amounts of the 3 proteins with dose-response effects, but only in the apical compartment. A lowered ELISA response was also observed when either IL-6, IL-8 or MCP-1 were mixed with punicalagin in a cell-free culture medium, indicating a direct molecular interaction. In conclusion, the punicalagin-rich PomH extract tested showed anti-inflammatory properties in the Caco-2 in vitro intestinal model. It acted both on the pro-inflammatory gene transcription and protein levels, the later phenomenon being possibly due to a direct molecular trapping. These data suggest that pomegranate husk could be an interesting natural source contributing to prevent intestinal chronic inflammation.

  11. Development of Caco-2 cells co-expressing CYP3A4 and NADPH-cytochrome P450 reductase using a human artificial chromosome for the prediction of intestinal extraction ratio of CYP3A4 substrates.

    Science.gov (United States)

    Takenaka, Toru; Kazuki, Kanako; Harada, Naomoto; Kuze, Jiro; Chiba, Masato; Iwao, Takahiro; Matsunaga, Tamihide; Abe, Satoshi; Oshimura, Mitsuo; Kazuki, Yasuhiro

    2017-02-01

    The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers. The metabolic activity of CYP3A4 (midazolam 1'-hydroxylase activity) in the CYP3A4-CPR-HAC/Caco-2 cells was constant from 22 to 35 passages, indicating that HAC vectors conferred sufficient and sustained CYP3A4 activity to CYP3A4-CPR-HAC/Caco-2 cells. The strong relationship between the metabolic extraction ratios (ER) obtained from the CYP3A4-CPR-HAC/Caco-2 cells and calculated intestinal extraction ratios in humans (Eg) from reported intestinal availability (Fg) was found for 17 substrates of CYP3A4 (r2 = 0.84). The present study suggests that the CYP3A4-CPR-HAC/Caco-2 cell monolayer can serve as an in vitro tool that facilitates the prediction of intestinal extraction ratio (or availability) in humans. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  12. Drug permeation across intestinal epithelial cells using porous silicon nanoparticles.

    Science.gov (United States)

    Bimbo, Luis M; Mäkilä, Ermei; Laaksonen, Timo; Lehto, Vesa-Pekka; Salonen, Jarno; Hirvonen, Jouni; Santos, Hélder A

    2011-04-01

    Mesoporous silicon particles hold great potential in improving the solubility of otherwise poorly soluble drugs. To effectively translate this feature into the clinic, especially via oral or parenteral administration, a thorough understanding of the interactions of the micro- and nanosized material with the physiological environment during the delivery process is required. In the present study, the behaviour of thermally oxidized porous silicon particles of different sizes interacting with Caco-2 cells (both non-differentiated and polarized monolayers) was investigated in order to establish their fate in a model of intestinal epithelial cell barrier. Particle interactions and TNF-α were measured in RAW 264.7 macrophages, while cell viabilities, reactive oxygen species and nitric oxide levels, together with transmission electron microscope images of the polarized monolayers, were assessed with both the Caco-2 cells and RAW 264.7 macrophages. The results showed a concentration and size dependent influence on cell viability and ROS-, NO- and TNF-α levels. There was no evidence of the porous nanoparticles crossing the Caco-2 cell monolayers, yet increased permeation of the loaded poorly soluble drug, griseofulvin, was shown. Copyright © 2010 Elsevier Ltd. All rights reserved.

  13. Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates.

    Science.gov (United States)

    DiMarco, Rebecca L; Hunt, Daniel R; Dewi, Ruby E; Heilshorn, Sarah C

    2017-06-01

    The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2 cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2 cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported in vivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by

  14. Transport and uptake effects of marine complex lipid liposomes in small intestinal epithelial cell models.

    Science.gov (United States)

    Du, Lei; Yang, Yu-Hong; Xu, Jie; Wang, Yu-Ming; Xue, Chang-Hu; Kurihara, Hideyuki; Takahashi, Koretaro

    2016-04-01

    Nowadays, marine complex lipids, including starfish phospholipids (SFP) and cerebrosides (SFC) separated from Asterias amurensis as well as sea cucumber phospholipids (SCP) and cerebrosides (SCC) isolated from Cucumaria frondosa, have received much attention because of their potent biological activities. However, little information is known on the transport and uptake of these lipids in liposome forms in small intestinal cells. Therefore, this study was undertaken to investigate the effects of these complex lipid liposomes on transport and uptake in Caco-2 and M cell monolayer models. The results revealed that SFP and SCP contained 42% and 47.9% eicosapentaenoic acid (EPA), respectively. The average particle sizes of liposomes prepared in this study were from 169 to 189 nm. We found that the transport of the liposomes across the M cell monolayer model was much higher than the Caco-2 cell monolayer model. The liposomes consisting of SFP or SCP showed significantly higher transport and uptake than soy phospholipid (soy-PL) liposomes in both Caco-2 and M cell monolayer models. Our results also exhibited that treatment with 1 mM liposomes composed of SFP or SCP for 3 h tended to increase the EPA content in phospholipid fractions of both differentiated Caco-2 and M cells. Moreover, it was also found that the hybrid liposomes consisting of SFP/SFC/cholesterol (Chol) revealed higher transport and uptake across the M cell monolayer in comparison with other liposomes. Furthermore, treatment with SFP/SFC/Chol liposomes could notably decrease the trans-epithelial electrical resistance (TEER) values of Caco-2 and M cell monolayers. The present data also showed that the cell viability of differentiated Caco-2 and M cells was not affected after the treatment with marine complex lipids or soy-PL liposomes. Based on the data in this study, it was suggested that marine complex lipid liposomes exhibit prominent transport and uptake in small intestinal epithelial cell models.

  15. Extraction of Antioxidant Components from Bidens pilosa Flowers and Their Uptake by Human Intestinal Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Charng-Cherng Chyau

    2013-01-01

    Full Text Available Bidens pilosa L. var. radiata (BPR, Asteraceae is a commonly used folk medicine for treating various disorders such as diabetes, inflammation and hypertension. Recent studies to determine its chemical composition have revealed three di-O-caffeoylquinic acids (DiCQAs and three polyacetylene glucosides (PGAs to be among the major bioactive markers. To obtain the major compounds of these two chemical classes, the ethyl acetate fraction (EM obtained using liquid-liquid partition from the methanol extract resulted in a fraction with the highest total phenolic and total flavonoid contents and antioxidant activities in radical scavenging and ferric reducing power assays. To assess the bioavailability of EM, we examined the in vitro uptake using the Caco-2 human colonic cell line. The apparent permeability coefficient (Papp for each of the compounds within PGAs measured in both apical (AP to basolateral (BL and BL to AP was found to preferentially appear BL to AP direction, indicated that a basolateral to apical efflux system was detected in the study. DiCQAs had a lower efflux ratio than those from PGAs (2.32–3.67 vs. 6.03–78.36. Thus, it strongly implies that most of the DiCQAs are better absorbed than the PGAs.

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

    Science.gov (United States)

    Drummond, Coyne G; Nickerson, Cheryl A; Coyne, Carolyn B

    2016-01-01

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

  17. Exogenous HIV-1 Nef Upsets the IFN-γ-Induced Impairment of Human Intestinal Epithelial Integrity

    Science.gov (United States)

    Quaranta, Maria Giovanna; Vincentini, Olimpia; Felli, Cristina; Spadaro, Francesca; Silano, Marco; Moricoli, Diego; Giordani, Luciana; Viora, Marina

    2011-01-01

    Background The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line. Methodology/Principal Findings We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepitelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade. Conclusion/Significance Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions. PMID:21858117

  18. Exogenous HIV-1 Nef upsets the IFN-γ-induced impairment of human intestinal epithelial integrity.

    Directory of Open Access Journals (Sweden)

    Maria Giovanna Quaranta

    Full Text Available The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepithelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions.

  19. A novel in vitro co-culture model comprised of Caco-2/RBL-2H3 cells to evaluate anti-allergic effects of food factors through the intestine.

    Science.gov (United States)

    Yamashita, Sae; Yokoyama, Yuki; Hashimoto, Takashi; Mizuno, Masashi

    2016-08-01

    The prevalence of type I allergic diseases such as food allergy and allergic rhinitis has increased. Therefore, many studies have focused on food factors with anti-allergic activities in recent years. In order to investigate the effect of food factors on mast cell activation, a RBL-2H3 cell monoculture system has been widely used, in which various food factors have been reported to inhibit degranulation of RBL-2H3 cells. However, some orally administered food factors do not interact directly with immune cells but do so indirectly through intestinal epithelial cells. In this report, we established a novel in vitro co-culture model to evaluate anti-allergic effects of orally administered food factors. The co-culture system, comprised of Caco-2 cells (apical component) and RBL-2H3 cells (basolateral component), was able to evaluate the effects of two flavonoids that are known to have the inhibitory effects on mast cell degranulation. Moreover, we evaluated the anti-allergic effects of Enterococcus faecalis strains that are not absorbed through the intestine. We identified two strains of lactic acid bacteria that had inhibitory effects on mast cell degranulation using this co-culture system and possessed anti-allergic properties in a passive cutaneous anaphylaxis model mouse. This novel in vitro co-culture model was applicable for finding food factors with anti-allergic effects and might be useful for examining its anti-allergic mechanisms. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Modified Dietary Fiber from Cassava Pulp and Assessment of Mercury Bioaccessibility and Intestinal Uptake Using an In Vitro Digestion/Caco-2 Model System.

    Science.gov (United States)

    Kachenpukdee, Natta; Santerre, Charles R; Ferruzzi, Mario G; Oonsivilai, Ratchadaporn

    2016-07-01

    The ability of modified dietary fiber (MDF) generated from cassava pulp to modulate the bioaccessibility and intestinal absorption of heavy metals may be helpful to mitigate health risk associated with select foods including select fish high in methyl mercury. Using a coupled in vitro digestion/Caco-2 human intestinal cell model, the reduction of fish mercury bioaccessibility and intestinal uptake by MDF was investiaged. MDF was prepared from cassava pulp, a byproduct of tapioca production. The highest yield (79.68%) of MDF was obtained by enzymatic digestion with 0.1% α-amylase (w/v), 0.1% amyloglucosidase (v/v) and 1% neutrase (v/v). MDF and fish tissue were subjected to in vitro digestion and results suggest that MDF may reduce mercury bioaccessibility from fish to 34% to 85% compared to control in a dose-dependent manner. Additionally, accumulation of mercury from digesta containing fish and MDF was only modestly impacted by the presence of MDF. In conclusion, MDF prepared from cassava pulp may be useful as an ingredient to reduce mercury bioavailability from food such as fish specifically by inhibiting mercury transfer to the bioaccessibile fraction during digestion. © 2016 Institute of Food Technologists®

  1. Evaluation of the intestinal transport of a phenylethanoid glycoside-rich extract from Cistanche deserticola across the Caco-2 cell monolayer model.

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    Yuan Gao

    Full Text Available Phenylethanoid glycosides (PhGs, a class of polyphenolic compounds, are considered one of major bioactive constituents of Cistanche deserticola Y.C. Ma (CD, whose extract is orally used in traditional Chinese medicine. Although previous pharmacological studies have reported that PhGs exert many activities, their intestinal transport profiles have not been clarified. In this study, we investigated the intestinal permeability of a PhG-rich extract (PRE from CD as an integrated system in the Caco-2 cell monolayer model using a bioassay system. The results showed that PRE is primarily transported via poorly absorbed passive diffusion down a concentration gradient without efflux, which provides the pharmacokinetic basis for the clinical application of PhGs in CD. We also determined the intestinal permeability of three major PhGs [acteoside (AC, isoacteoside (IS and echinacoside (EC] by HLPC. Furthermore, we developed a novel HPLC-fluorescence detection method to accurately determine the flux amount of AC and IS. As expected, the transport characteristics of the three PhGs are consistent with those of PRE, indicating that the present bioassay system is appropriate and reliable for the evaluation of the transport characteristics of active ingredient groups (AIG in PRE. Moreover, this system may also be suitable for other plant extracts given appropriate bioactivity.

  2. Evaluation of the intestinal transport of a phenylethanoid glycoside-rich extract from Cistanche deserticola across the Caco-2 cell monolayer model.

    Science.gov (United States)

    Gao, Yuan; Zong, Chuanjie; Liu, Fen; Fang, Lei; Cai, Runlan; Shi, Yue; Chen, Xi; Qi, Yun

    2015-01-01

    Phenylethanoid glycosides (PhGs), a class of polyphenolic compounds, are considered one of major bioactive constituents of Cistanche deserticola Y.C. Ma (CD), whose extract is orally used in traditional Chinese medicine. Although previous pharmacological studies have reported that PhGs exert many activities, their intestinal transport profiles have not been clarified. In this study, we investigated the intestinal permeability of a PhG-rich extract (PRE) from CD as an integrated system in the Caco-2 cell monolayer model using a bioassay system. The results showed that PRE is primarily transported via poorly absorbed passive diffusion down a concentration gradient without efflux, which provides the pharmacokinetic basis for the clinical application of PhGs in CD. We also determined the intestinal permeability of three major PhGs [acteoside (AC), isoacteoside (IS) and echinacoside (EC)] by HLPC. Furthermore, we developed a novel HPLC-fluorescence detection method to accurately determine the flux amount of AC and IS. As expected, the transport characteristics of the three PhGs are consistent with those of PRE, indicating that the present bioassay system is appropriate and reliable for the evaluation of the transport characteristics of active ingredient groups (AIG) in PRE. Moreover, this system may also be suitable for other plant extracts given appropriate bioactivity.

  3. Phosphatidylcholine passes through lateral tight junctions for paracellular transport to the apical side of the polarized intestinal tumor cell-line CaCo2.

    Science.gov (United States)

    Stremmel, Wolfgang; Staffer, Simone; Gan-Schreier, Hongying; Wannhoff, Andreas; Bach, Margund; Gauss, Annika

    2016-09-01

    Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1±6.3nmolh(-1)mMPC(-1) was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl(-) and HCO3(-) through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Sensory active piperine analogues from Macropiper excelsum and their effects on intestinal nutrient uptake in Caco-2 cells.

    Science.gov (United States)

    Obst, Katja; Lieder, Barbara; Reichelt, Katharina V; Backes, Michael; Paetz, Susanne; Geißler, Katrin; Krammer, Gerhard; Somoza, Veronika; Ley, Jakob P; Engel, Karl-Heinz

    2017-03-01

    The phytochemical profile of Macropiper excelsum (G.Forst.) Miq. subsp. excelsum (Piperaceae), a shrub which is widespread in New Zealand, was investigated by LC-MS-guided isolation and characterization via HR-ESI-TOF-MS and NMR spectroscopy. The isolated compounds were sensorily evaluated to identify their contribution to the overall taste of the crude extract with sweet, bitter, herbal and trigeminal impressions. Besides the known non-volatile Macropiper compounds, the lignans (+)-diayangambin and (+)-excelsin, four further excelsin isomers, (+)-diasesartemin, (+)-sesartemin, (+)-episesartemin A and B were newly characterized. Moreover, piperine and a number of piperine analogues as well as trans-pellitorine and two homologues, kalecide and (2E,4E)-tetradecadienoic acid N-isobutyl amide were identified in M. excelsum, some of them for the first time. Methyl(2E,4E)-7-(1,3-benzodioxol-5-yl)hepta-2,4-dienoate was identified and characterized for the first time in nature. Sensory analysis of the pure amides indicated that they contributed to the known chemesthetic effects of Macropiper leaves and fruits. Since the pungent piperine has been shown to affect glucose and fatty acid metabolism in vivo in previous studies, piperine itself and four of the isolated compounds, piperdardine, chingchengenamide A, dihydropiperlonguminine, and methyl(2E,4E)-7-(1,3-benzodioxol-5-yl)hepta-2,4-dienoate, were investigated regarding their effects on glucose and fatty acid uptake by enterocyte-like Caco-2 cells, in concentrations ranging from 0.1 to 100 μM. Piperdardine showed the most pronounced effect, with glucose uptake increased by 83 ± 18% at 100 μM compared to non-treated control cells. An amide group seems to be advantageous for glucose uptake stimulation, but not necessarily for fatty acid uptake-stimulating effects of piperine-related compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Effects of prefermented cereals or the end products of fermentation on growth and metabolism of enterocyte-like Caco-2 cells and on intestinal health of restrictedly fed weanling pigs.

    Science.gov (United States)

    Bruininx, E M A M; Koninkx, J F J G; Binnendijk, G P; Zandstra, T; Heetkamp, M J W; van der Peet-Schwering, C M C; Gerrits, W J J

    2010-01-01

    To unravel the underlying mechanisms that explain the positive effects of prefermented cereals on in vivo gastrointestinal (GI) architecture and function, an in vitro experiment using a human small intestinal epithelial cell model (Caco-2) was performed. A range of dilutions (0% to 10%) of the supernatants of three liquid experimental diets, as well as Na-lactate were used in an in vitro experiment to assess their effect on cellular growth, metabolism, differentiation and mucosal integrity using Caco-2. The experimental diets contained, in addition to a protein rich basal diet (60%), (1) a liquid control diet (C) containing 40% of a mixture of barley and wheat (ratio 3 : 1) or (2) a liquid diet (F) containing 40% prefermented barley and wheat or (3) C with the addition of the fermentation end-products (organic acids and ethanol) in concentrations similar to those in the fermented diet (FP). For F, the mixture of barley and wheat was fermented at 35°C for 48 h. Parallel to the in vitro experiment, 18 groups of eight weanling pigs were assigned to one of the experimental diets during a 14-day in vivo experiment. Each group was fed restrictively. The results of the in vitro experiment showed that the lowest dose of both F- and FP-supernatants had no clear effects on the cell proliferation, but incubation with 5% and 10% of the F- and FP-supernatants decreased the cell numbers at day 19. DNA, RNA, protein and glycoprotein synthesis in differentiated Caco-2 cells were stimulated by incubation with the lower concentrations (0.5% to 2.5%) of F- and FP-supernatants whereas the higher concentrations (5% and 10%) had no effect. Both the F- and FP-supernatants decreased the specific sucrase-isomaltase activity in a dose-dependent manner, but the effects on the specific aminopeptidase activities were less clear. Mucosal integrity initially decreased after incubation with the highest F- and FP-supernatants and started to recover between 24 and 48 h. The results of the in vivo

  6. Polyphenol-rich black chokeberry (Aronia melanocarpa) extract regulates the expression of genes critical for intestinal cholesterol flux in Caco-2 cells.

    Science.gov (United States)

    Kim, Bohkyung; Park, Youngki; Wegner, Casey J; Bolling, Bradley W; Lee, Jiyoung

    2013-09-01

    Black chokeberry (Aronia melanocarpa) is a rich source of polyphenols. The hypolipidemic effects of polyphenol-rich black chokeberry extract (CBE) have been reported, but underlying mechanisms have not been well characterized. We investigated the effect of CBE on the expression of genes involved in intestinal lipid metabolism. Caco-2 cells were incubated with 50 or 100 μg/ml of CBE for 24 h for quantitative realtime polymerase chain reaction analysis. Expression of genes for cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase and sterol regulatory element binding protein 2), apical cholesterol uptake (Niemann-Pick C1 Like 1 and scavenger receptor class B Type 1) and basolateral cholesterol efflux [ATP-binding cassette transporter A1 (ABCA1)] was significantly decreased by CBE compared with control. Western blot analysis confirmed that CBE inhibited expression of these proteins. In contrast, CBE markedly induced mRNA and/or protein levels of ABCG5 and ABCG8 that mediate apical cholesterol efflux to the intestinal lumen. Furthermore, CBE significantly increased mRNA and protein levels of low-density lipoprotein (LDL) receptor, and cellular LDL uptake. Expression of genes involved in lipid metabolism and lipoprotein assembly, including sterol regulatory element-binding protein 1c, fatty acid synthase and acyl-CoA oxidase 1, was significantly decreased by CBE in a dose-dependent manner. Concomitantly, CBE significantly increased sirtuin 1, 3 and 5 mRNA levels, while it decreased SIRT-2. Our data suggest that hypolipidemic effects of CBE may be attributed, at least in part, to increased apical efflux of LDL-derived cholesterol and to decreased chylomicron formation in the intestine; and specific isoforms of SIRT may play an important role in this process. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Bile acid receptor TGR5 overexpression is associated with decreased intestinal mucosal injury and epithelial cell proliferation in obstructive jaundice.

    Science.gov (United States)

    Ji, Chen-Guang; Xie, Xiao-Li; Yin, Jie; Qi, Wei; Chen, Lei; Bai, Yun; Wang, Na; Zhao, Dong-Qiang; Jiang, Xiao-Yu; Jiang, Hui-Qing

    2017-04-01

    Bile acids stimulate intestinal epithelial proliferation in vitro. We sought to investigate the role of the bile acid receptor TGR5 in the protection of intestinal epithelial proliferation in obstructive jaundice. Intestinal tissues and serum samples were obtained from patients with malignant obstructive jaundice and from bile duct ligation (BDL) rats. Intestinal permeability and morphological changes in the intestinal mucosa were observed. The functions of TGR5 in cell proliferation in intestinal epithelial injury were determined by overexpression or knockdown studies in Caco-2 and FHs 74 Int cells pretreated with lipopolysaccharide (LPS). Internal biliary drainage was superior to external biliary drainage in recovering intestinal permeability and mucosal histology in patients with obstructive jaundice. In BDL rats, feeding of chenodeoxycholic acid (CDCA) decreased intestinal mucosa injury. The levels of PCNA, a marker of proliferation, increased in response to CDCA feeding and were paralleled by elevated TGR5 expression. CDCA upregulated TGR5 expression and promoted proliferation in Caco-2 and FHs 74 Int cells pretreated with LPS. Overexpression of TGR5 resulted in increased PCNA, cell viability, EdU incorporation, and the proportion of cells in S phase, whereas knockdown of TGR5 had the opposite effect. Our data indicate that bile acids promote intestinal epithelial cell proliferation and decrease mucosal injury by upregulating TGR5 expression in obstructive jaundice. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells.

    Science.gov (United States)

    Roubos-van den Hil, P J; Nout, M J R; Beumer, R R; van der Meulen, J; Zwietering, M H

    2009-03-01

    This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action. Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells. Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds. The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases.

  9. Bioaccessibility, biotransformation, and transport of alpha-mangostin from Garcinia mangostana (Mangosteen) using simulated digestion and Caco-2 human intestinal cells.

    Science.gov (United States)

    Bumrungpert, Akkarach; Kalpravidh, Ruchaneekorn W; Suksamrarn, Sunit; Chaivisuthangkura, Apinya; Chitchumroonchokchai, Chureeporn; Failla, Mark L

    2009-05-01

    alpha- and gamma-Mangostin are the most abundant prenylated xanthones present in the fruit of the mangosteen tree. These compounds have been reported to possess numerous bioactivities that have provided the impetus for use of mangosteen products as nutraceuticals and in functional foods and dietary supplements. The health-promoting benefits of mangosteen are dependent on delivery of the xanthones to target tissues. Here, we used simulated digestion and Caco-2 cells to investigate the digestive stability, bioaccessibility, and intestinal cell transport of alpha- and gamma- mangostin. Recovery of alpha- and gamma-mangostin after simulated digestion of pericarp and fruit pulp exceeded 90%. Transfer of alpha- and gamma-mangostin to the aqueous fraction during simulated digestion was efficient (65-74%) and dependent on bile salts suggesting that micellarization is required for optimal bioaccessibility of xanthones. Cell uptake of xanthones from micelles was dose dependent and intracellular concentrations were maximum by 1 h. Both free and phase II metabolites of alpha-mangostin were transported in the basolateral compartment and metabolites also effluxed into the apical chamber. Transepithelial transport of alpha-mangostin was increased during prandial-like compared to fasted conditions suggesting that absorption is enhanced by dietary fat.

  10. Comparison of ceftibuten transport across Caco-2 cells and rat jejunum mounted on modified Ussing chambers.

    Science.gov (United States)

    Menon, R M; Barr, W H

    2003-10-01

    Ceftibuten uptake into Caco-2 cells and intestinal brush border membrane vesicles is mediated by the dipeptide transport system (PEPT1). The apical to basolateral transport characteristics of ceftibuten across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was examined. Mannitol was used as a paracellular marker along with trans-epithelial electrical resistance (TEER) for monitoring tight junction permeability. Transport across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was linear across the concentration range 0.25-10 mM. The net flux of mannitol and ceftibuten was higher across rat jejunum compared with Caco-2 cells. At a donor concentration of 0.25 mM, ceftibuten transport across Caco-2 cells was found to be pH dependent. Glycyl proline, a dipeptide, and 2,4- dinitrophenol, an energy poison, caused a reduction in the permeability of 0.25 mM ceftibuten across Caco-2 cells. Benzoic acid and adipic acid also inhibited transcellular transport of ceftibuten. At a donor concentration of 0.25 mM, passive paracellular transport accounts for about 60% and the active carrier mediated mechanism accounts for about 40% of ceftibuten transport across Caco-2 cells. None of the inhibitors however, had a significant effect on ceftibuten transport across rat jejunum mounted on a modified Ussing chamber at a donor concentration of 0.25 mM. In the concentration range 0.25-10 mM, ceftibuten is predominantly transported by paracellular mechanisms across rat jejunum and a mixture of active and passive transport across Caco-2 cells. Copyright 2003 John Wiley & Sons, Ltd.

  11. Degradation of the transcription factors NF-κB, STAT3, and STAT5 is involved in Entamoeba histolytica-induced cell death in Caco-2 colonic epithelial cells.

    Science.gov (United States)

    Kim, Kyeong Ah; Min, Arim; Lee, Young Ah; Shin, Myeong Heon

    2014-10-01

    Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-κB (p65) in Caco-2 cells. However, IκB, an inhibitor of NF-κB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-κB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-κB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

  12. Intestinal anti-inflammatory activity of lentinan: influence on IL-8 and TNFR1 expression in intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Yosuke Nishitani

    Full Text Available Inflammatory bowel disease (IBD is characterized by chronic inflammation of the gastrointestinal tract. It is unknown whether β-1,3;1,6-glucan can induce immune suppressive effects. Here, we study intestinal anti-inflammatory activity of Lentinula edodes-derived β-1,3;1,6-glucan, which is known as lentinan. Dextran sulfate sodium (DSS-induced colitis mice were used to elucidate effects of lentinan in vivo. In the cellular level assessment, lentinan was added into a co-culture model consisting of intestinal epithelial Caco-2 cells and LPS-stimulated macrophage RAW264.7 cells. Ligated intestinal loop assay was performed for assessing effects of lentinan on intestinal epithelial cells (IECs in vivo. Oral administration of lentinan (100 µg/mouse significantly ameliorated DSS-induced colitis in body weight loss, shortening of colon lengths, histological score, and inflammatory cytokine mRNA expression in inflamed tissues. Lentinan reduced interleukin (IL-8 mRNA expression and nuclear factor (NF-κB activation in Caco-2 cells without decreasing of tumor necrosis factor (TNF-α production from RAW264.7 cells. Flow cytometric analysis revealed that surface levels of TNF receptor (TNFR 1 were decreased by lentinan treatment. A clathrin-mediated endocytosis inhibitor, monodansylcadaverine, canceled lentinan inhibition of IL-8 mRNA expression. Moreover, lentinan inhibited TNFR1 expression in Caco-2 cells in both protein and mRNA level. Lentinan also inhibited TNFR1 mRNA expression in mouse IECs. These results suggest that lentinan exhibits intestinal anti-inflammatory activity through inhibition of IL-8 mRNA expression associated with the inhibition of NF-κB activation which is triggered by TNFR1 endocytosis and lowering of their expression in IECs. Lentinan may be effective for the treatment of gut inflammation including IBD.

  13. Commensal microbiota-induced microRNA modulates intestinal epithelial permeability through the small GTPase ARF4.

    Science.gov (United States)

    Nakata, Kazuaki; Sugi, Yutaka; Narabayashi, Hikari; Kobayakawa, Tetsuro; Nakanishi, Yusuke; Tsuda, Masato; Hosono, Akira; Kaminogawa, Shuichi; Hanazawa, Shigemasa; Takahashi, Kyoko

    2017-09-15

    The intestinal tract contains many commensal bacteria that modulate various physiological host functions. Dysbiosis of commensal bacteria triggers dysfunction of the intestinal epithelial barrier, leading to the induction or aggravation of intestinal inflammation. To elucidate whether microRNA plays a role in commensal microbiome-dependent intestinal epithelial barrier regulation, we compared transcripts in intestinal epithelial cells (IECs) from conventional and germ-free mice and found that commensal bacteria induced the expression of miR-21-5p in IECs. miR-21-5p increased intestinal epithelial permeability and up-regulated ADP ribosylation factor 4 (ARF4), a small GTPase, in the IEC line Caco-2. We also found that ARF4 expression was up-regulated upon suppression of phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4), which are known miR-21-5p targets, by RNAi. Furthermore, ARF4 expression in epithelial cells of the large intestine was higher in conventional mice than in germ-free mice. ARF4 suppression in the IEC line increased the expression of tight junction proteins and decreased intestinal epithelial permeability. These results indicate that commensal microbiome-dependent miR-21-5p expression in IECs regulates intestinal epithelial permeability via ARF4, which may therefore represent a target for preventing or managing dysfunction of the intestinal epithelial barrier. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Permeation of Insulin, Calcitonin and Exenatide across Caco-2 Monolayers: Measurement Using a Rapid, 3-Day System

    Science.gov (United States)

    Gupta, Vivek; Doshi, Nishit; Mitragotri, Samir

    2013-01-01

    Objectives Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules. However, the conventional Caco-2 monolayer model has several drawbacks including labor-intensive culture process, unphysiological growth conditions, lack of reproducibility and limited throughput. Here, we report on the use of 3-day Caco-2 monolayers for assessing permeability of polypeptide drugs. Methods The 3-day monolayers were grown in a commercially available transwell set-up, which facilitates rapid development of the Caco-2 monolayers in an intestinal epithelial differentiation mimicking environment. This set-up included use of serum-free medium of defined composition with supplements such as butyric acid, hormones, growth factors, and other metabolites, reported to regulate the differentiation of intestinal epithelial cells in vivo. We measured permeability of 3 different therapeutic polypeptides; insulin, calcitonin, and exenatide across the monolayer. Results Preliminary validation of the monolayer was carried out by confirming dose-dependent permeation of FITC-insulin and sulforhodamine-B. Transport of insulin, calcitonin, and exenatide measured at different loading concentrations suggests that the permeability values obtained with 3-day cultures resemble more closely the values obtained with ex vivo models compared to permeability values obtained with conventional 21-day cultures. Conclusions Short-term 3-day Caco-2 monolayers provide new opportunities for developing reproducible and high-throughput models for screening of therapeutic macromolecules for oral absorption. PMID:23483881

  15. In vitro System for Assessing Dioxin Absorption by Intestinal Epithelial Cells and for Preventing this Absorption by Food Substances

    OpenAIRE

    Natsume, Yayoi; Satsu, Hideo; Hamada, Mika; Kitamura, Kazushige; Okamoto, Naoto; Shimizu, Makoto

    2005-01-01

    A system for assessing intestinal dioxin absorption was established by applying a Caco-2 cell monolayer and stable dioxin-responsive cell line. The stable dioxin-responsive cell line was established by introducing a plasmid incorporating the human CYP1A1 promoter into human hepatic HepG2 genomic DNA upstream of the luciferase gene. 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) was added to the apical side of differentiated human intestinal epithelial Caco-2 cell monolayers that had been cultured on...

  16. Histone code of genes induced by co-treatment with a glucocorticoid hormone agonist and a p44/42 MAPK inhibitor in human small intestinal Caco-2 cells.

    Science.gov (United States)

    Inamochi, Yuko; Mochizuki, Kazuki; Goda, Toshinao

    2014-01-01

    Inactivation of glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) is thought to be important in small intestinal maturation and expression of genes related to intestinal differentiation and functions. We investigated target genes induced by co-treatment for 48h with a glucocorticoid hormone agonist, dexamethasone (Dex), and a p44/42 MAPK inhibitor, PD98059 (PD), in a small intestine-like cell line (Caco-2) using microarray analysis. We also investigated whether expression changes of the target genes induced by the co-treatment are associated with histone modifications around these genes. Co-treatment of Caco-2 cells with Dex and PD enhanced several genes related to intestinal differentiation and functions such as SCNN1A, FXYD3, LCT and LOX. Induction of the SCNN1A gene was associated with increased presence of acetylated histone H3 and H4 and di-methylated histone H3 at lysine (K) 4 around the transcribed region of the gene, and induction of the FXYD3 gene was associated with increased presence of acetylated histones H3 and H4 from the promoter/enhancer to the transcribed region of the gene. Induction of LCT and LOX genes was associated with increased presence of acetylated histone H4 on the promoter/enhancer region of the genes. Histone acetylation and/or histone H3 K4 methylation around the promoter/enhancer or/and transcribed regions of target genes are associated with induction of the genes by co-treatment with Dex and PD in Caco-2 cells. The histone code is specific to each gene with respect to induction by glucocorticoid hormone and inhibition of p44/42 MAPK in Caco-2 cells. © 2013.

  17. Probiotic activity of Enterococcus faecalis CECT7121: effects on mucosal immunity and intestinal epithelial cells.

    Science.gov (United States)

    Castro, M S; Molina, M A; Azpiroz, M B; Díaz, A M; Ponzio, R; Sparo, M D; Manghi, M A; Canellada, A M

    2016-10-01

    To analyse the effect of Enterococcus faecalis CECT7121 on intestinal epithelial cells (IECs) and its effects on the mucosal immune response. Enterococcus faecalis CECT7121 showed a high adhesion capacity to completely and heterogeneously differentiated human intestinal epithelial cell line (Caco-2 cells). In addition, the contact of this bacterium with Caco-2 cells did not induce inflammatory chemokines (IL-8 and CCL-20). The presence of IgA(+) and IL-6(+) cells in the small intestine, as well as the production of inflammatory cytokines (TNFα, IL-6 and IL-12) in the gut, was determined after intragastric inoculation of Ent. faecalis CECT7121 in BALB/c mice. The administration of Ent. faecalis CECT7121 increased the number of IgA(+) cells in the intestinal lamina propria without modifying the percentage of IL-6(+) cells. No differences were observed in the cytokines measured in the intestinal extracts between probiotic-treated and control mice. Enterococcus faecalis CECT7121 stimulates local mucosal immunity and adheres to IECs without inducing inflammatory signals. Our results indicate that, apart from its already reported systemic immune activity, Ent. faecalis CECT7121 has a modulatory effect at a local level. © 2016 The Society for Applied Microbiology.

  18. Interaction of cruciferin-based nanoparticles with Caco-2 cells and Caco-2/HT29-MTX co-cultures.

    Science.gov (United States)

    Akbari, Ali; Lavasanifar, Afsaneh; Wu, Jianping

    2017-12-01

    The objective of this work was to assess the potential of Cruciferin/Calcium (Cru/Ca) and Cruciferin/Chitosan (Cru/Cs) nanoparticles for oral drug delivery. For this purpose, Cru/Ca and Cru/Cs nanoparticles were developed through cold gelation of Cruciferin, a major canola protein, and in interaction with calcium and chitosan, respectively. The extent and rate of particle uptake in Caco-2 cells and Caco-2/HT29 co-culture was then evaluated by fluorescence spectroscopy as well as flow cytometry. Through pre-incubation of Caco-2 cell monolayer with specific endocytosis inhibitors, the mechanism of cell uptake was investigated. Our results showed that the uptake of negatively-charged Cru/Ca particles to be ∼3 times higher than positively-charged Cru/Cs ones by Caco-2 cells. Presence of mucus secreted by HT29 cells in their co-culture with Caco-2 had negligible influence on the uptake and transport of both particles. In contrast to Cru/Ca particles which were dissociated in the simulated gastrointestinal conditions, digestion of Cru/Cs particles resulted in 6- and 2-fold increase in the cellular uptake and transport of encapsulated coumarin in the latter particles, respectively. While the presence of mucus in Caco-2/HT29 co-culture caused 40-50% decrease of cellular uptake and transport for coumarin encapsulated in digested Cru/Cs particles, it had no significant effect on the cell uptake and transport of coumarin associated with Cru/Ca particles after digestion. Energy-dependent mechanisms were the dominant mechanism for uptake of both undigested and digested particles. Therefore, in Caco-2/HT29 co-culture which closely simulated intestinal epithelial cells, undigested Cru/Ca and Cru/Cs particles had the ability to penetrate mucus layers, while digested Cru/Cs particles showed mucoadhesive property, and digested Cru/Ca particles were dissociated. Our results points to a potential for cruciferin based nanoparticles for oral drug delivery. The long-term objective of

  19. Intestinal epithelial dysplasia (tufting enteropathy

    Directory of Open Access Journals (Sweden)

    de Serres Natacha

    2007-04-01

    Full Text Available Abstract Intestinal epithelial dysplasia (IED, also known as tufting enteropathy, is a congenital enteropathy presenting with early-onset severe intractable diarrhea causing sometimes irreversible intestinal failure. To date, no epidemiological data are available, however, the prevalence can be estimated at around 1/50,000–100,000 live births in Western Europe. The prevalence seems higher in areas with high degree of consanguinity and in patients of Arabic origin. Infants develop within the first days after birth a watery diarrhea persistent in spite of bowel rest and parenteral nutrition. Some infants are reported to have associated choanal rectal or esophageal atresia. IED is thought to be related to abnormal enterocytes development and/or differentiation. Nonspecific punctuated keratitis was reported in more than 60% of patients. Histology shows various degree of villous atrophy, with low or without mononuclear cell infiltration of the lamina propria but specific histological abnormalities involving the epithelium with disorganization of surface enterocytes with focal crowding, resembling tufts. Several associated specific features were reported, including abnormal deposition of laminin and heparan sulfate proteoglycan (HSPG in the basement membrane, increased expression of desmoglein and ultrastructural changes in the desmosomes, and abnormal distribution of α2β1 integrin adhesion molecules. One model of transgenic mice in which the gene encoding the transcription factor Elf3 is disrupted have morphologic features resembling IED. Parental consanguinity and/or affected siblings suggest an autosomal recessive transmission but the causative gene(s have not been yet identified making prenatal diagnosis unavailable. Some infants have a milder phenotype than others but in most patients, the severity of the intestinal malabsorption even with enteral feeding make them totally dependent on daily long-term parenteral nutrition with a subsequent

  20. Characterization of in vitro effects of patulin on intestinal epithelial and immune cells.

    Science.gov (United States)

    Assunção, R; Alvito, P; Kleiveland, C R; Lea, T E

    2016-05-27

    The intestinal mucosa is the first biological barrier encountered by natural toxins, and could possibly be exposed to high amounts of dietary mycotoxins. Patulin (PAT), a mycotoxin produced by Penicillium spp. during fruit spoilage, is one of the best known enteropathogenic mycotoxins able to alter functions of the intestine (Maresca et al., 2008). This study evaluated the effects of PAT on barrier function of the gut mucosa utilizing the intestinal epithelial cell model Caco-2, and scrutinized immunomodulatory effects using human peripheral blood mononuclear cells (PBMC) and human blood monocyte-derived dendritic cells (moDCs) as test systems. PAT exposure reduced Caco-2 cell viability at concentrations above 12μM. As expected, the integrity of a polarized Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50μM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50μM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain 2 (MLC2) declined. Also, levels of phospho-MLC2 (p-MLC2) increased after 24h of exposure to 50μM of PAT. T cell proliferation was highly sensitive to PAT with major effects for concentrations above 10nM of PAT. The same conditions did not affect the maturation of moDC. PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and the phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of moDC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. The proton-coupled amino acid transporter hPAT1 is the main transporter involved in vigabatrin uptake in intestinal Caco-2 cells

    DEFF Research Database (Denmark)

    Nøhr, Martha Kampp; Hansen, Steen Honore'; Brodin, Birger

    2012-01-01

    transporter hPAT1. The aim of the project was to identify if transporters are involved in cellular uptake of vigabatrin in Caco-2 cells. Methods: The uptake rate of vigabatrin was measured in Caco-2 cells at pH 6.0 or 7.4 for 15 min after application of 0.1 – 25.0 mM vigabatrin. The inhibitory effect...... of selected amino acids and -derivatives on the apical vigabatrin uptake in Caco-2 cells was investigated. Vigabatrin samples were analyzed using liquid chromatography (LC) coupled to a mass selective detector (MSD). Results: The uptake rate of vigabatrin in Caco-2 cells was pH-dependent. The uptake...... of vigabatrin was saturable at pH 6.0 with a Michaelis constant, Km of 12.7 ± 3.7 mM and a maximal flux, Jmax of 3.7 ± 0.5 nmol•min-1•cm-2. The presences of hPAT1 ligands significantly inhibited the uptake of vigabatrin in Caco-2 cells at pH 6.0, whereas hPAT1 non-ligands did not. Discussion: The saturability...

  2. Lycium barbarum L. Polysaccharide (LBP Reduces Glucose Uptake via Down-Regulation of SGLT-1 in Caco2 Cell

    Directory of Open Access Journals (Sweden)

    Huizhen Cai

    2017-02-01

    Full Text Available Lycium barbarum L. polysaccharide (LBP is prepared from Lycium barbarum L. (L. barbarum, which is a traditional Chinese medicine. LPB has been shown to have hypoglycemic effects. In order to gain some mechanistic insights on the hypoglycemic effects of LBP, we investigated the uptake of LBP and its effect on glucose absorption in the human intestinal epithelial cell line Caco2 cell. The uptake of LBP through Caco2 cell monolayer was time-dependent and was inhibited by phloridzin, a competitive inhibitor of SGLT-1. LPB decreased the absorption of glucose in Caco2 cell, and down-regulated the expression of SGLT-1. These results suggest that LBP might be transported across the human intestinal epithelium through SGLT-1 and it inhibits glucose uptake via down-regulating SGLT-1.

  3. Vitamin D differentially regulates Salmonella-induced intestine epithelial autophagy and interleukin-1β expression.

    Science.gov (United States)

    Huang, Fu-Chen

    2016-12-21

    To investigate the effects of active vitamin D3 on autophagy and interleukin (IL)-1β expression in Salmonella-infected intestinal epithelial cells (IECs). Caco-2 cells, NOD2 siRNA-, Atg16L1 siRNA- or vitamin D receptor (VDR) siRNA-transfected Caco-2 cells were pretreated with 1,25-dihydroxyvitamin D3 (1,25D3), and then infected by wild-type S. typhimurium strain SL1344. The conversion of LC3-I to LC3-II was detected by Western blot analysis and LC3+ autophagosome was analyzed by immunofluorescence. Caco-2 cells or VDR siRNA-transfected cells were pretreated with 1,25D3, and then infected by SL1344. Membrane protein and total RNA were analyzed by Western blot and RT-PCR for VDR and Atg16L1 protein and mRNA expression, respectively. Atg16L1 siRNA-transfected Caco-2 cells were pretreated by 1,25D3 and then infected with SL1344. Total RNA was analyzed by RT-PCR for IL-1β mRNA expression. The active form of vitamin D, 1,25D3, showed enhanced VDR-mediated Atg16L1 mRNA expression, membranous Atg16L1 protein expression leading to enhanced autophagic LC3II protein expression and LC3 punctae in Salmonella-infected Caco-2 cells which was counteracted by Atg16L1 and VDR siRNA, but Atg16L1 mediated suppression of IL-1β expression. Thus, active vitamin D may enhance autophagy but suppress inflammatory IL-1β expression in Salmonella-infected IECs. Active vitamin D might enhance autophagic clearance of Salmonella infection, while modulation of inflammatory responses prevents the host from detrimental effects of overwhelming inflammation.

  4. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells.

    Science.gov (United States)

    Bhattacharya, Sujoy; Ray, Ramesh M; Johnson, Leonard R

    2014-03-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.

  5. The dynamic expression of the epidermal growth factor receptor and epidermal growth factor ligand family in a differentiating intestinal epithelial cell line.

    Science.gov (United States)

    Kuwada, S K; Li, X F; Damstrup, L; Dempsey, P J; Coffey, R J; Wiley, H S

    1999-01-01

    The Caco-2 intestinal epithelial cell line differentiates when cultured on plastic or permeable filters, and offers a valuable system to study events associated with enterocytic differentiation in vitro. Little is known as to whether the expression of the epidermal growth factor receptor (EGFR) and its ligands changes as intestinal epithelial cells differentiate. We found that total cellular EGFR protein and mRNA transcript levels were relatively unchanged during Caco-2 cell differentiation, but the expression of surface EGFR and patterns of steady state epidermal growth factor (EGF)-family ligand expression changed significantly. EGFR affinity, surface EGFR expression levels, and the repertoire of expressed EGF-family ligands, were different between Caco-2 cells cultured on plastic and filters. Functionally, EGFR-mediated cell proliferation and tyrosine phosphorylation of the signal transduction protein SHC could be inhibited in Caco-2 cells cultured on filters, but not on plastic. Thus, the substrate on which the cells were grown and the degree of cell differentiation strongly modulate EGFR affinity, EGFR surface expression, the steady state expression of EGF-family ligands, as well as, EGFR-mediated cellular responses. Our results suggest that the EGFR system is regulated during intestinal epithelial cell differentiation primarily at the level of ligand expression.

  6. Protective effect of carboxytmethylpachymaran on TNF-α-induced damage in Caco-2 cell monolayers.

    Science.gov (United States)

    Zhang, Jinwei; Lu, Yue; Wei, Jianan; Li, Li; Han, Ling

    2016-12-01

    This study was carried out to study the protective effect of carboxytmethylpachymaran (CMP) on TNF-α-induced intestinal epithelial barrier dysfunction in Caco-2 cell monolayers and the underlying mechanism. The Caco-2 cell monolayers were pretreated with 50, 100 or 150μg/mL CMP for 72h, and then they were exposed to 100ng/mL tumor necrosis factor alpha (TNF-α) for 72h. The results showed that CMP alleviated the drop of trans-epithelial electrical resistance (TEER) and the increase of phenol red flux induced by TNF-α. CMP also ameliorated TNF-α-induced decrease of mRNA/protein expression and distribution of Occludin and ZO-3 which were chosen as makers of tight junction (TJ). Additionally, the increased protein expressions of MLCK, phosphorylation level of myosin light chain (p-MLC), NF-κB p-P65 and p-IκBα induced by TNF-α were significantly inhibited by CMP. This study demonstrates the protective effect of CMP on TNF-α-induced damage of intestinal epithelial barrier in Caco-2 monolayers and discovers that the suppression of MLCK-p-MLC signaling regulated by NF-κB might be one of the mechanisms underlying the protective effect of CMP. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Synergistic Effects of Zinc Oxide Nanoparticles and Fatty Acids on Toxicity to Caco-2 Cells

    DEFF Research Database (Denmark)

    Cao, Yi; Roursgaard, Martin; Kermanizadeh, Ali

    2015-01-01

    Fatty acids exposure may increase sensitivity of intestinal epithelial cells to cytotoxic effects of zinc oxide (ZnO) nanoparticles (NPs). This study evaluated the synergistic effects of ZnO NPs and palmitic acid (PA) or free fatty acids (FFAs) mixture (oleic/PA 2:1) on toxicity to human colon...... epithelial (Caco-2) cells. The ZnO NPs exposure concentration dependently induced cytotoxicity to Caco-2 cells showing as reduced proliferation and activity measured by 3 different assays. PA exposure induced cytotoxicity, and coexposure to ZnO NPs and PA showed the largest cytotoxic effects. The presence...... of FFAs mixture did not affect the ZnO NPs-induced cytotoxicity. Filtration of freshly prepared suspension of NPs through a 0.45-µm pore size membrane significantly reduced the cytotoxicity, indicating a role of concentration or size of particles in cytotoxic effects. The ZnO NPs and PA coexposure induced...

  8. Transport Mechanisms of Solid Lipid Nanoparticles across Caco-2 Cell Monolayers and their Related Cytotoxicology.

    Science.gov (United States)

    Chai, Gui-Hong; Xu, Yingke; Chen, Shao-Qing; Cheng, Bolin; Hu, Fu-Qiang; You, Jian; Du, Yong-Zhong; Yuan, Hong

    2016-03-09

    Solid lipid nanoparticles (SLNs) have been extensively investigated and demonstrated to be a potential nanocarriers for improving oral bioavailability of many drugs. However, the molecular mechanisms related to this discovery are not yet understood. Here, the molecular transport mechanisms of the SLNs crossing simulative intestinal epithelial cell monolayers (Caco-2 cell monolayers) were studied. The cytotoxicology results of the SLNs in Caco-2 cells demonstrated that the nanoparticles had low cytotoxicity, had no effect on the integrity of the cell membrane, did not induce oxidative stress, and could significantly reduce cell membrane fluidity. The endocytosis of the SLNs was time-dependent, and their delivery was energy-dependent. For the first time, the transport of the SLNs was directly verified to be a vesicle-mediated process. The internalization of the SLNs was mediated by macropinocytosis pathway and clathrin- and caveolae (or lipid raft)-related routes. Transferrin-related endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus were confirmed to be the main destinations of the SLNs in Caco-2 cells. As for the transport of the SLNs in Caco-2 cell monolayers, the results demonstrated that the SLNs transported to the basolateral side were intact, and the transport of the nanoparticles did not destroy the structure of tight junctions. The transcytosis of the SLNs across the Caco-2 cell monolayer was demonstrated to be mediated by the same routes as that in the endocytosis study. The ER, Golgi apparatus, and microtubules were confirmed to be important for the transport of the SLNs to both the basolateral and apical membrane sides. This study provides a more thoroughly understand of SLNs transportation crossing intestinal epithelial cell monolayers and could be beneficial for the fabrication of SLNs.

  9. Acute Effects of Sugars and Artificial Sweeteners on Small Intestinal Sugar Transport: A Study Using CaCo-2 Cells As an In Vitro Model of the Human Enterocyte.

    Science.gov (United States)

    O'Brien, Patrick; Corpe, Christopher Peter

    2016-01-01

    The gastrointestinal tract is responsible for the assimilation of nutrients and plays a key role in the regulation of nutrient metabolism and energy balance. The molecular mechanisms by which intestinal sugar transport are regulated are controversial. Based on rodent studies, two models currently exist that involve activation of the sweet-taste receptor, T1R2/3: an indirect model, whereby luminal carbohydrates activate T1R2/3 expressed on enteroendocrine cells, resulting in the release of gut peptides which in turn regulate enterocyte sugar transport capacity; and a direct model, whereby T1R2/3 expressed on the enterocyte regulates enterocyte function. To study the direct model of intestinal sugar transport using CaCo-2 cells, a well-established in vitro model of the human enterocyte. Uptake of 10mM 14C D-Glucose and D-Fructose into confluent CaCo-2/TC7 cells was assessed following 3hr preincubation with sugars and artificial sweeteners in the presence and absence of the sweet taste receptor inhibitor, lactisole. Expression of the intestinal sugar transporters and sweet-taste receptors were also determined by RT-PCR. In response to short term changes in extracellular glucose and glucose/fructose concentrations (2.5mM to 75mM) carrier-mediated sugar uptake mediated by SGLT1 and/or the facilitative hexose transporters (GLUT1,2,3 and 5) was increased. Lactisole and artificial sweeteners had no effect on sugar transport regulated by glucose alone; however, lactisole increased glucose transport in cells exposed to glucose/fructose. RT-PCR revealed Tas1r3 and SGLT3 gene expression in CaCo-2/TC7 cells, but not Tas1r2. In the short term, enterocyte sugar transport activities respond directly to extracellular glucose levels, but not fructose or artificial sweeteners. We found no evidence of a functional heterodimeric sweet taste receptor, T1R2/3 in CaCo-2 cells. However, when glucose/fructose is administered together there is an inhibitory effect on glucose transport

  10. Acute Effects of Sugars and Artificial Sweeteners on Small Intestinal Sugar Transport: A Study Using CaCo-2 Cells As an In Vitro Model of the Human Enterocyte.

    Directory of Open Access Journals (Sweden)

    Patrick O'Brien

    Full Text Available The gastrointestinal tract is responsible for the assimilation of nutrients and plays a key role in the regulation of nutrient metabolism and energy balance. The molecular mechanisms by which intestinal sugar transport are regulated are controversial. Based on rodent studies, two models currently exist that involve activation of the sweet-taste receptor, T1R2/3: an indirect model, whereby luminal carbohydrates activate T1R2/3 expressed on enteroendocrine cells, resulting in the release of gut peptides which in turn regulate enterocyte sugar transport capacity; and a direct model, whereby T1R2/3 expressed on the enterocyte regulates enterocyte function.To study the direct model of intestinal sugar transport using CaCo-2 cells, a well-established in vitro model of the human enterocyte.Uptake of 10mM 14C D-Glucose and D-Fructose into confluent CaCo-2/TC7 cells was assessed following 3hr preincubation with sugars and artificial sweeteners in the presence and absence of the sweet taste receptor inhibitor, lactisole. Expression of the intestinal sugar transporters and sweet-taste receptors were also determined by RT-PCR.In response to short term changes in extracellular glucose and glucose/fructose concentrations (2.5mM to 75mM carrier-mediated sugar uptake mediated by SGLT1 and/or the facilitative hexose transporters (GLUT1,2,3 and 5 was increased. Lactisole and artificial sweeteners had no effect on sugar transport regulated by glucose alone; however, lactisole increased glucose transport in cells exposed to glucose/fructose. RT-PCR revealed Tas1r3 and SGLT3 gene expression in CaCo-2/TC7 cells, but not Tas1r2.In the short term, enterocyte sugar transport activities respond directly to extracellular glucose levels, but not fructose or artificial sweeteners. We found no evidence of a functional heterodimeric sweet taste receptor, T1R2/3 in CaCo-2 cells. However, when glucose/fructose is administered together there is an inhibitory effect on glucose

  11. Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression.

    Directory of Open Access Journals (Sweden)

    Akihiro Watari

    Full Text Available Several stressors are known to influence epithelial tight junction (TJ integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM and ataxia telangiectasia mutated and Rad3-related protein (ATR, and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.

  12. Lactobacillus protects the integrity of intestinal epithelial barrier damaged by pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Qinghua eYu

    2015-03-01

    Full Text Available Pathogens invade intestinal mucosal barrier through phagocytosis of antigen presenting cells (dendritic cell, microfold cells, or through the invasion into the intestinal epithelial directly. Some pathogens could damage the cell junction between epithelial cells and use the paracellular pathway as an entrance to invade. Moreover, some Lactobacillus could inhibit the adhesion of the pathogens and protect the integrity of the cell junction and mucosal barrier. This research focused on the potential therapeutic effect of Lactobacillus fructosus (L. fructosus C2 to attenuate ETEC K88 or S. typhimurium SL1344 induced changes to mucosal barrier. The results demonstrated that treatment of polarized Caco-2 cells with L. fructosus C2 reduced the permeation of dextran, and expression of IL-8, p-ERK and p-JNK when cells were infected with pathogenic bacteria. The findings indicated that L. fructosus C2 exerted a protective effect against the damage to the integrity of Caco-2 cells by ETEC or S. typhimurium infection.

  13. Co-incubation of PMN and CaCo-2 cells modulates inflammatory potential.

    Science.gov (United States)

    Schaefer, M B; Schaefer, C A; Hecker, M; Morty, R E; Witzenrath, M; Seeger, W; Mayer, K

    2017-05-20

    Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We examined interactions of PMN and intestinal epithelial cell-like CaCo-2 cells to elucidate their regulation of inflammatory signalling and the impact of cyclooxygenase (COX), nitric oxide (NO) and platelet-activating factor (PAF). Human PMN and CaCo-2 cells, separately and in co-incubation, were stimulated with the calcium ionophore A23187 or with N-Formyl-methionyl-leucyl-phenylalanin (fMLP) that activates PMN only. Human neutrophil elastase (HNE) and respiratory Burst were measured. To evaluate the modulation of inflammatory crosstalk we applied inhibitors of COX (acetyl salicylic acid; ASA), NO-synthase (N-monomethyl-L-arginin; L-NMMA), and the PAF-receptor (WEB2086). Unstimulated, co-incubation of CaCo-2 cells and PMN led to significantly reduced Burst and elevated HNE as compared to PMN. After stimulation with A23187, co-incubation resulted in an inhibition of Burst and HNE. Using fMLP co-incubation failed to modulate Burst but increased HNE. Without stimulation, all three inhibitors abolished the effect of co-incubation on Burst but did not change HNE.  ASA partly prevented modulation of Burst L-NMMA and WEB2086 did not change Burst but abolished mitigation of HNE. Without stimulation, co-incubation reduced Burst and elevated HNE. Activation of PMN and CaCo-2 cells by fMLP as compared to A23187 resulted in a completely different pattern of Burst and HNE, possibly due to single vs. dual cell activation. Anti-inflammatory effect of co-incubation might in part be due to due to COX-signalling governing Burst whereas NO- and PAF-dependent signalling seemed to control HNE release.

  14. Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lihua; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Yang, Yang [Department of General Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Yang, Hua, E-mail: hwbyang@126.com [Department of General Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing 400037 (China)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. Black-Right-Pointing-Pointer The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. Black-Right-Pointing-Pointer GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. Black-Right-Pointing-Pointer GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

  15. Effect of Gelsemium elegans and Mussaenda pubescens, the Components of a Detoxification Herbal Formula, on Disturbance of the Intestinal Absorptions of Indole Alkaloids in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Yinghao Wang

    2017-01-01

    Full Text Available Gelsemium elegans (GE is a kind of well-known toxic plant. It can be detoxified by Mussaenda pubescens (MP, but the detoxification mechanism is still unclear. Thus, a detoxification herbal formula (GM comprising GE and MP was derived. The Caco-2 cells monolayer model was used to evaluate GM effects on transporting six kinds of indole alkaloids of GE. The bidirectional transport studies demonstrated that absorbance percentage of indole alkaloids in GE increased linearly over time. But in GM, Papp (AP→BL values of the most toxic members, gelsenicine, humantenidine, and gelsevirine, were lower than that of Papp (BL→AP (P<0.05. The prominent analgesic effect members, gelsemine and koumine, were approximately 1.00 in γ values. Nowhere was this increasing efflux more pronounced than in the case of indole alkaloids with N-O structure. In the presence of verapamil, the γ values of humantenidine, gelsenicine, gelsevirine, and humantenine were decreased by 43.69, 41.42, 36.00, and 8.90 percent, respectively. The γ values in presence of ciclosporin were homologous with a decrease of 42.32, 40.59, 34.00, and 15.07 percent. It suggested that the efflux transport was affected by transporters. Taken together, due to the efflux transporters participation, the increasing efflux of indole alkaloids from GM was found in Caco-2 cells.

  16. Estimating intestinal absorption of inorganic and organic selenium compounds by in vitro flux and biotransformation studies in Caco-2 cells and ICP-MS detection

    DEFF Research Database (Denmark)

    Gammelgaard, Bente; Rasmussen, Laura Hyrup; Gabel-Jensen, Charlotte

    2012-01-01

    The aim of the present work was to compare and estimate absorption and biotransformation of selected selenium compounds by studying their fluxes across Caco-2 cells. Five different selenium compounds, selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenate, selenite, and methylselen......The aim of the present work was to compare and estimate absorption and biotransformation of selected selenium compounds by studying their fluxes across Caco-2 cells. Five different selenium compounds, selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenate, selenite......) were also studied. Selenium absorptive and exsorptive fluxes and accumulation in cell cytosol were analyzed by means of flow injection inductively coupled plasma mass spectrometry (ICP-MS). Absorptive flux of SeMet, MeSeCys, and selenate showed values correlating to complete in vivo absorption, while......SeA, including volatile species, whereas no significant increases in fluxes were observed. In summary, the absorption of selenite selenate and the selenoamino acids is considered complete under physiological conditions, but the absorption mechanisms and metabolism of the compounds are different. © 2011 Springer...

  17. Acidic conditions exacerbate interferon-gamma-induced intestinal epithelial hyperpermeability: role of peroxynitrous acid.

    Science.gov (United States)

    Unno, N; Hodin, R A; Fink, M P

    1999-08-01

    Nitric oxide (NO*) derived from exogenous donors has been shown to increase the permeability of cultured intestinal epithelial monolayers, an effect that is augmented by mildly acidic conditions. Because interferon-gamma (IFN-gamma) also increases intestinal epithelial permeability, at least partly by an NO*-dependent mechanism, we sought to determine whether IFN-gamma-induced hyperpermeability is increased under acidic conditions. Human intestinal epithelial (Caco-2BBe) cells were grown as monolayers on permeable supports in bicameral chambers. Permeability was assessed by measuring transepithelial electrical resistance (TER) or the transepithelial passage of fluorescein disulfonic acid. Inducible nitric oxide synthase (iNOS) messenger RNA expression was determined by northern blot analysis. Concentrations of nitrite and nitrate (NO2-/NO3-), stable oxidation products of NO*, were determined using the Greiss reaction. Cellular adenosine triphosphate (ATP) levels were determined using the luciferin/luciferase method. Incubation of Caco-2BBe monolayers with INF-gamma (1000 units/mL) at an extracellular pH (pHo) of 7.4 increased permeability to fluorescein disulfonic acid and decreased TER. However, incubation of monolayers with IFN-gamma under mildly acidic conditions (i.e., pHo 7.0-6.6) accelerated the decrease in TER and augmented the increase in permeability induced by the cytokine. IFN-gamma-induced iNOS messenger RNA expression and NO2-/NO3- accumulation in medium were unaffected by acidic conditions. At pHo 7.4, incubation of Caco-2BBe monolayers with IFN-gamma (1000 units/mL) for 72 hrs had no effect on intracellular ATP content compared with monolayers simultaneously incubated under the same conditions but in the absence of the cytokine. However, when the cells were incubated for 72 hrs with the same concentration of IFN-gamma under mildly acidic conditions (i.e., pHo 7.0 or 6.6), ATP levels were significantly decreased. At pHo 7.0, IFN

  18. Bifidobacteria isolated from infants and cultured on human milk oligosaccharides affect intestinal epithelial function.

    Science.gov (United States)

    Chichlowski, Maciej; De Lartigue, Guillaume; German, J Bruce; Raybould, Helen E; Mills, David A

    2012-09-01

    Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in selected bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of selected bifidobacteria on HMO stimulates the intestinal epithelium. Caco-2 and HT-29 cells were incubated with lactose (LAC)- or HMO-grown Bifidobacterium longum subsp infantis (B infantis) or B bifidum. Bacterial adhesion and translocation were measured by real-time quantitative polymerase chain reaction. Expression of pro- and anti-inflammatory cytokines and tight junction proteins was analyzed by real-time reverse transcriptase. Distribution of tight junction proteins was measured using immunofluorescent microscopy. We showed that HMO-grown B infantis had a significantly higher rate of adhesion to HT-29 cells compared with B bifidum. B infantis also induced expression of a cell membrane glycoprotein, P-selectin glycoprotein ligand-1. Both B infantis and B bifidum grown on HMO caused less occludin relocalization and higher expression of anti-inflammatory cytokine, interleukin-10 compared with LAC-grown bacteria in Caco-2 cells. B bifidum grown on HMO showed higher expression of junctional adhesion molecule and occludin in Caco-2 cells and HT-29 cells. There were no significant differences between LAC or HMO treatments in bacterial translocation. The study provides evidence for the specific relation between HMO-grown bifidobacteria and intestinal epithelial cells. To our knowledge, this is the first study describing HMO-induced changes in the bifidobacteria-intestinal cells interaction.

  19. In vitro System for Assessing Dioxin Absorption by Intestinal Epithelial Cells and for Preventing this Absorption by Food Substances.

    Science.gov (United States)

    Natsume, Yayoi; Satsu, Hideo; Hamada, Mika; Kitamura, Kazushige; Okamoto, Naoto; Shimizu, Makoto

    2005-01-01

    A system for assessing intestinal dioxin absorption was established by applying a Caco-2 cell monolayer and stable dioxin-responsive cell line. The stable dioxin-responsive cell line was established by introducing a plasmid incorporating the human CYP1A1 promoter into human hepatic HepG2 genomic DNA upstream of the luciferase gene. 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) was added to the apical side of differentiated human intestinal epithelial Caco-2 cell monolayers that had been cultured on a semipermeable membrane. The basal medium was taken after an appropriate incubation time and added to the dioxin-responsive cells, the TCDD content then being analyzed by a luciferase assay. The amount of TCDD in the basal medium increased in a dose- and time-dependent manner, the results being sufficiently sensitive and reproducible. The inhibition of TCDD permeability to the Caco-2 cell monolayer by such food substances as chlorophyll, insoluble corn fiber and tea dregs were observed by this in vitro assessment system. The system will therefore be useful to identify food substances having a preventive effect on the intestinal absorption of dioxins.

  20. Chitosan nanoparticles reduce LPS-induced inflammatory reaction via inhibition of NF-κB pathway in Caco-2 cells.

    Science.gov (United States)

    Tu, Jue; Xu, Yinglei; Xu, Jianqin; Ling, Yun; Cai, Yueqin

    2016-05-01

    Chitosan nanoparticles (CNP), an extensively oral-administered drug carrier, was investigated for the anti-inflammatory effects on LPS-inflamed Caco-2 cells and the relate mechanisms. CNP could alleviate the decrease of transepithelial electrical resistance (TEER) induced by LPS in Caco-2 monolayer, and significantly inhibit LPS-induced production of TNF-α, MIF, IL-8 and MCP-1 in a dose-dependent manner. PCR array assay revealed that CNP down-regulated the mRNA expression levels of TLR4 in LPS-inflamed Caco-2 cells. CNP was further showed to reduce cytoplasmic IκB-α degradation and nuclear NF-κB p65 levels in LPS-inflamed Caco-2 cells. These results suggested that CNP suppressed LPS-induced inflammatory response by decreasing permeability of intestinal epithelial monolayer and secretion of pro-inflammatory cytokine in Caco-2 cells, which were partially mediated by NF-κB signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Commensal-derived OMVs elicit a mild proinflammatory response in intestinal epithelial cells.

    Science.gov (United States)

    Patten, Daniel A; Hussein, Enas; Davies, Scott P; Humphreys, Paul N; Collett, Andrew

    2017-05-01

    Under normal physiological conditions, the intestinal immunity remains largely hyporesponsive to the commensal microbiota, yet also retains the inherent ability to rapidly respond to pathogenic antigens. However, immunomodulatory activities of extracellular products from commensal bacteria have been little studied, with previous investigations generally utilizing the live bacterium to study microbiota-epithelial interactions. In this study, we demonstrate that extracellular products of a commensal bacterium, Escherichia coli C25, elicit a moderate release of proinflammatory IL-8 and stimulate transcriptional up-regulation of Toll-like receptors (TLRs) in intestinal epithelial cell lines HT29-19A and Caco-2. Additionally, we show that removal of outer membrane vesicles (OMVs) reduces the proinflammatory effect of secreted products from E. coli C25. Furthermore, we show that isolated OMVs have a dose-dependent proinflammatory effect on intestinal epithelial cells (IECs). Interestingly, a relatively high concentration (40 µg ml-1 protein) of OMVs had no significant regulatory effects on TLR mRNA expression in both cell lines. Finally, we also demonstrate that pre-incubation with E. coli C25-derived OMVs subsequently inhibited the internalization of the bacterium itself in both cell lines. Taken together, our results suggest that commensal-derived extracellular products, in particular OMVs, could significantly contribute to intestinal homeostasis. We also demonstrate a unique interaction between commensal-derived OMVs and host cells.

  2. Protective effects of ψ taraxasterol 3-O-myristate and arnidiol 3-O-myristate isolated from Calendula officinalis on epithelial intestinal barrier.

    Science.gov (United States)

    Dall'Acqua, Stefano; Catanzaro, Daniela; Cocetta, Veronica; Igl, Nadine; Ragazzi, Eugenio; Giron, Maria Cecilia; Cecconello, Laura; Montopoli, Monica

    2016-03-01

    The triterpene esters ᴪ taraxasterol-3-O-myristate (1) and arnidiol-3-O-myristate (2) were tested for their ability to protect epithelial intestinal barrier in an in vitro model. Their effects on ROS production and on trans-epithelial resistance were investigated on CaCo-2 cell monolayers both in basal and stress-induced conditions. Both compounds were able to modulate the stress damage induced by H2O2 and INFγ+TNFα, showing a potential use as model compounds for the study of new therapeutic agents for intestinal inflammations. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Role of protein tyrosine kinase in the effect of IP6 on IL-8 secretion in intestinal epithelial cells.

    Science.gov (United States)

    Wawszczyk, Joanna; Orchel, Arkadiusz; Kapral, Małgorzata; Wéglarz, Ludmiła

    2013-01-01

    Phytic acid (IP6) is a major fiber-associated component of a diet physiologically present in human intestines. Studies showed that this phytochemical can modulate immune functions of intestinal epithelium through regulation of proinflammatory cytokines secretion but mechanisms underlying these cellular response to IP6 have weakly been examined, as yet. The aim of this study was to determine the role of protein tyrosine kinase (PTK) in secretion of IL-8, a central proinflammatory cytokine, by unstimulated and IL-1beta-stimulated intestinal epithelial cells Caco-2 treated with IP6 (1 and 2.5 mM). To study the involvement of PTK signal pathway in IL-8 secretion, inhibitors of phosphotyrosine phosphatase (sodium orthovanadate, OV) and tyrosine kinase (genistein, GEN) were incubated with Caco-2 cells prior to IP6 treatment. IP6 had suppressive effect on basal and IL-1beta-stimulated IL-8 secretion by cells. The effect of OV on IL-8 release by cells treated with IP6 was different under constitutive and stimulated conditions. Secretion of IL-8 was significantly down-regulated in cells with GEN and GEN plus IP6 treatment. In addition, total PTK activity in both unstimulated and IL-1beta stimulated cells was determined in the presence of IP6. The results suggest that physiological intestinal concentrations of IP6 may have an inhibitory effect on IL-8 secretion by Caco-2 cells and one of the mechanisms of its action is the inhibition of PTK signaling cascade. The study revealed for the first time that PTKs could be one of the molecular targets for IP6 effects in the intestinal epithelial cells.

  4. Epithelial Cell Damage Activates Bactericidal/Permeability Increasing-Protein (BPI Expression in Intestinal Epithelium

    Directory of Open Access Journals (Sweden)

    Arjun Balakrishnan

    2017-08-01

    Full Text Available As the first line of defense against invading pathogen, intestinal epithelium produces various antimicrobial proteins (AMP that help in clearance of pathogen. Bactericidal/permeability-increasing protein (BPI is a 55 kDa AMP that is expressed in intestinal epithelium. Dysregulation of BPI in intestinal epithelium is associated with various inflammatory diseases like Crohn’s Disease, Ulcerative colitis, and Infectious enteritis’s. In this paper, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S. aureus infection and pore-forming toxins (Streptolysin and Listeriolysin. Cells detect changes in potassium level as a Danger-associated molecular pattern associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that the BPI expression in murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium and Shigella flexneri whereas mutants that do not cause intestinal damage (STM ΔfliC and STM ΔinvC did not induce BPI expression. Our results suggest that epithelial damage associated with infection act as a signal to induce BPI expression.

  5. Brucella invasion of human intestinal epithelial cells elicits a weak proinflammatory response but a significant CCL20 secretion.

    Science.gov (United States)

    Ferrero, Mariana C; Fossati, Carlos A; Rumbo, Martín; Baldi, Pablo C

    2012-10-01

    In spite of the frequent acquisition of Brucella infection by the oral route in humans, the interaction of the bacterium with cells of the intestinal mucosa has been poorly studied. Here, we show that different Brucella species can invade human colonic epithelial cell lines (Caco-2 and HT-29), in which only smooth species can replicate efficiently. Infection with smooth strains did not produce a significant cytotoxicity, while the rough strain RB51 was more cytotoxic. Infection of Caco-2 cells or HT-29 cells with either smooth or rough strains of Brucella did not result in an increased secretion of TNF-α, IL-1β, MCP-1, IL-10 or TGF-β as compared with uninfected controls, whereas all the infections induced the secretion of IL-8 and CCL20 by both cell types. The MCP-1 response to flagellin from Salmonella typhimurium was similar in Brucella-infected or uninfected cells, ruling out a bacterial inhibitory mechanism as a reason for the weak proinflammatory response. Infection did not modify ICAM-1 expression levels in Caco-2 cells, but increased them in HT-29 cells. These results suggest that Brucella induces only a weak proinflammatory response in gut epithelial cells, but produces a significant CCL20 secretion. The latter may be important for bacterial dissemination given the known ability of Brucella to survive in dendritic cells. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. Identification of multi-drug resistant Pseudomonas aeruginosa clinical isolates that are highly disruptive to the intestinal epithelial barrier

    Directory of Open Access Journals (Sweden)

    Shevchenko Olga

    2006-06-01

    Full Text Available Abstract Background Multi-drug resistant Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide. In this study, we focused on the virulence of multi-drug resistant clinical strains P. aeruginosa against the intestinal epithelial barrier, since P. aeruginosa can cause lethal sepsis from within the intestinal tract of critically ill and immuno-compromised patients via mechanisms involving disruption of epithelial barrier function. Methods We screened consecutively isolated multi-drug resistant P. aeruginosa clinical strains for their ability to disrupt the integrity of human cultured intestinal epithelial cells (Caco-2 and correlated these finding to related virulence phenotypes such as adhesiveness, motility, biofilm formation, and cytotoxicity. Results Results demonstrated that the majority of the multi-drug resistant P. aeruginosa clinical strains were attenuated in their ability to disrupt the barrier function of cultured intestinal epithelial cells. Three distinct genotypes were found that displayed an extreme epithelial barrier-disrupting phenotype. These strains were characterized and found to harbor the exoU gene and to display high swimming motility and adhesiveness. Conclusion These data suggest that detailed phenotypic analysis of the behavior of multi-drug resistant P. aeruginosa against the intestinal epithelium has the potential to identify strains most likely to place patients at risk for lethal gut-derived sepsis. Surveillance of colonizing strains of P. aeruginosa in critically ill patients beyond antibiotic sensitivity is warranted.

  7. Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells.

    Science.gov (United States)

    Lebeer, Sarah; Claes, Ingmar; Tytgat, Hanne L P; Verhoeven, Tine L A; Marien, Eyra; von Ossowski, Ingemar; Reunanen, Justus; Palva, Airi; Vos, Willem M de; Keersmaecker, Sigrid C J De; Vanderleyden, Jos

    2012-01-01

    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid.

  8. Effect of water-soluble fraction of cherry tomatoes on the adhesion of probiotics and Salmonella to intestinal epithelial cells.

    Science.gov (United States)

    Koh, Jong Ho; Kim, Narae; Hwang, Dahyun; Lim, Young-Hee

    2013-12-01

    Tomato is one of the most consumed vegetables in the world and contains many valuable nutritional components. Here we investigate the prebiotic effects of cherry tomatoes for improving gut health. Water-soluble dietary fiber was prepared from fresh and processed (heat treatment at 80 °C for 15 min) cherry tomato samples, each with and without Viscozyme L treatment. In the adhesion assays, all water-soluble dietary fiber samples improved adhesion of probiotics (Lactobacillus rhamnosus and Bifidobacterium bifidum) to intestinal epithelial cells (Caco-2 cells). Heat treatment in the preparation of juice from cherry tomatoes showed no significant effect on the adhesion of probiotics to Caco-2 cells. The oligofructose content of samples affected the intestinal adhesion of probiotic bacteria, with higher oligosaccharide concentrations associated with greater adhesion of probiotics and more inhibition of the adhesion of pathogens to Caco-2 cells. The present results suggest that cherry tomato can act as a prebiotic, with oligofructose potentially being one of its major prebiotic components. © 2013 Society of Chemical Industry.

  9. Uptake of label-free graphene oxide by Caco-2 cells is dependent on the cell differentiation status.

    Science.gov (United States)

    Kucki, Melanie; Diener, Liliane; Bohmer, Nils; Hirsch, Cordula; Krug, Harald F; Palermo, Vincenzo; Wick, Peter

    2017-06-21

    Understanding the interaction of graphene-related materials (GRM) with human cells is a key to the assessment of their potential risks for human health. There is a knowledge gap regarding the potential uptake of GRM by human intestinal cells after unintended ingestion. Therefore the aim of our study was to investigate the interaction of label-free graphene oxide (GO) with the intestinal cell line Caco-2 in vitro and to shed light on the influence of the cell phenotype given by the differentiation status on cellular uptake behaviour. Internalisation of two label-free GOs with different lateral size and thickness by undifferentiated and differentiated Caco-2 cells was analysed by scanning electron microscopy and transmission electron microscopy. Semi-quantification of cells associated with GRM was performed by flow cytometry. Undifferentiated Caco-2 cells showed significant amounts of cell-associated GRM, whereas differentiated Caco-2 cells exhibited low adhesion of GO sheets. Transmission electron microscopy analysis revealed internalisation of both applied GO (small and large) by undifferentiated Caco-2 cells. Even large GO sheets with lateral dimensions up to 10 µm, were found internalised by undifferentiated cells, presumably by macropinocytosis. In contrast, no GO uptake could be found for differentiated Caco-2 cells exhibiting an enterocyte-like morphology with apical brush border. Our results show that the internalisation of GO is highly dependent on the cell differentiation status of human intestinal cells. During differentiation Caco-2 cells undergo intense phenotypic changes which lead to a dramatic decrease in GRM internalisation. The results support the hypothesis that the cell surface topography of differentiated Caco-2 cells given by the brush border leads to low adhesion of GO sheets and sterical hindrance for material uptake. In addition, the mechanical properties of GRM, especially flexibility of the sheets, seem to be an important factor for

  10. Intact penetratin metabolite permeates across Caco-2 monolayers

    DEFF Research Database (Denmark)

    Birch, Ditlev; Christensen, Malene Vinther; Stærk, Dan

    . Previous studies have demonstrated that cell-penetrating peptides (CPPs) may be used as carriers in order to improve the bioavailability of a therapeutic cargo like insulin after oral administration. Penetratin, a commonly used CPP, has been shown to increase the uptake of insulin across Caco-2 cell......, the aim of the present study was to investigate penetratin metabolites with respect to effects on cellular viability, their epithelial permeation and cell uptake. Methods Extracellular and intracellular degradation of penetratin was assessed by incubation of the carrier peptide on the apical side of Caco......-2 cells cultured on permeable filter inserts and in cell lysates, respectively. The epithelial permeation of penetratin and the formed metabolites was assessed by using Caco-2 monolayers cultured on permeable filter inserts. Results Preliminary data revealed that at least one specific metabolite...

  11. Effect of rhamnolipids on permeability across Caco-2 cell monolayers.

    Science.gov (United States)

    Wallace, Charity J; Medina, Scott H; ElSayed, Mohamed E H

    2014-04-01

    This report describes the effect of rhamnolipids (RLs), an amphiphilic biosurfactant produced by the bacterium Pseudomonas aeruginosa, on the integrity and permeability across Caco-2 cell monolayers. We measured the trans-epithelial electrical resistance (TEER) and permeability of [(14)C]mannitol across Caco-2 cell monolayers upon incubation with 0.01-5.0% v/v RLs as a function of incubation time (30, 60, 90, and 120 min). We also studied the recovery of RL-treated Caco-2 cell monolayers upon incubation with Kaempferol, which is a natural flavonoid that promotes the assembly of the tight junctions. TEER of Caco-2 cell monolayers incubated with 0.01-5.0% v/v RLs solution dropped to 80-28% of that of untreated cells. Decline in TEER was associated with an increase in [(14)C]mannitol permeability as a function of RLs concentration and incubation time with Caco-2 cells. Incubation of RLs-treated Caco-2 cell monolayers with normal culture medium for 48 h did not restore barrier integrity. Whereas, incubation of a RLs-treated Caco-2 cells with culture medium containing Kaempferol for 24 h restored barrier function indicated by the higher TEER and lower [(14)C]mannitol permeability values. These results show the ability of RLs to modulate the integrity and permeability of Caco-2 cell monolayers in a concentration- and time-dependent fashion, which suggest their potential to function as a non-toxic permeation enhancer.

  12. [Effect of eicosapentaenoic acid on mRNA expression of tight junction protein ZO-1 in intestinal epithelial cells after Escherichia coli LF82 infection].

    Science.gov (United States)

    Hao, Li-Jun; Lin, Yan; Zhang, Wei; Tian, Jiao; Wang, Ya; Chen, Peng-De; Hu, Chong-Kang; Zeng, Ling-Chao; Yang, Jie; Wang, Bao-Xi; Jiang, Xun

    2017-06-01

    To investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection. The Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant. After EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (Pcoli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (Pcoli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (Pcoli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2

  13. Comparison of Caco-2, IEC-18 and HCEC cell lines as a model for intestinal absorption of genistein, daidzein and their glycodides

    NARCIS (Netherlands)

    Steensma, A.; Noteborn, H.P.J.M.; Kuiper, H.A.

    2004-01-01

    Genistein and daidzein receive much attention because of their potential to prevent hormone-related cancer and cardiovascular diseases. Limited information is available on the pharmacokinetics of these compounds like, for instance, intestinal uptake by humans and systematic bioavailability. In this

  14. The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: role in colorectal cancer progression.

    Directory of Open Access Journals (Sweden)

    Marie-Josée Langlois

    2010-12-01

    Full Text Available The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function, migration (wound assay, invasion (matrigel-coated transwells and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.

  15. Intestinal transport: studies with isolated epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kimmich, G.A.

    1979-12-01

    Isolated intestinal epithelial cells have been extremely useful for characterizing the nature of intestinal absorption processes and for providing insight into the energetics of Na/sup +/-dependent transport systems. This report describes a number of experimental approaches which have been used for investigating the specific epithelial transport systems involved in sugar absorption, but provides information which ultimately should prove useful for characterizing a number of different intestinal transport events. Similar experiments should also prove useful for exploring the effect of environmental agents on the function of intestinal tissue. In the case of sugars, net absorption is accomplished via a mucosal, Na/sup +/-dependent concentrative transport system acting in sequence with a passive serosal system which does not require Na/sup +/. The serosal system limits the full gradient-forming capability of the muscosal system. Agents such as phloretin or cytochalasin B which inhibit serosal transport allow the cells to establish sugar gradients as high as 70 fold in contrast to 10 to 15 fold gradients observed for control cells. Sevety-fold sugar gradients cannot be explained in terms of the energy available in the electrochemical potential for Na/sup +/ if the Na/sub 2/: sugar coupling stoichiometry is 1:1 as commonly assumed. New information indicates that the true Na/sup +/:sugar stoichiometry is in fact 2:1. Flow of two Na/sup +/ ions per sugar molecule down the transmembrane electrochemical potential for Na/sup +/ provides more than sufficient energy to account for observed 70 fold sugar gradients. If flow of sugar by other routes could be completely inhibited, theoretical sugar gradients as high as 400 could be achieved assuming that the cells maintain a membrane potential of -36 mV as measured for intact tissue.

  16. Absorption properties of micellar lipid metabolites into Caco2 cells.

    Science.gov (United States)

    Tsuzuki, Wakako

    2007-07-01

    To elucidate the absorption characteristics of dietary lipids in the human intestine, we investigated the cellular uptake of lipid metabolites using a differential monolayer of the Caco2 cells. As lipid metabolites, several free fatty acids and 2-monoacylglycerols, were formed a mixed micelle by bile salts and lysophospholipids and they were supplied to the Caco2 cells. To estimate the effect of the mixed micelles on the permeability of cells' membranes during incubation with the mixed micelles, the transepitherial electrical resistance (TEER) value was monitored, and no pronounced changes of TEER was detected. This suggested that mixed micelles did not affect their cellular properties of the barrier measured by TEER. The lipid metabolites transferred from the mixed micelle into the Caco2 cells were determined quantitatively by an enzymatic colorimetric method and were done by thin layer chromatography (TLC) for a species of acylglycerols. These highly sensitive methods enabled us to monitor the transepithelial transports of various kinds of non-isotope-labeled various lipid metabolites. Newly re-synthesized triacylglycerols were accumulated in Caco2 cells after 30 min incubation with the mixed micelles, and their amounts increased gradually for 4 h. The secretion of re-esterified triacylglycerols into a basolateral medium from the Caco2 cells began at 2 h after the mixed micelles were added to the apical medium. The intake of external lipid metabolites by the Caco2 cells were evaluated by an initial 2-h incubation with the mixed micelles. For example, 2-monomyristin and 2-monopalmitin were more rapidly transferred into the Caco2 cells from the mixed micelles than 2-monocaprin was. On the other hand, the absorption rates of capric acid, lauric acid and myristic acid by the cells were larger than those of stearic acid and oleic acid. It revealed that the side-chain structure of these lipid metabolites affected their absorption by the Caco2 cells. The results of this

  17. Increased production of the ether-lipid platelet-activating factor in intestinal epithelial cells infected by Salmonella enteritidis.

    Science.gov (United States)

    Egea, Laia; Giménez, Rosa; Lúcia, David; Modolell, Ines; Badía, Josefa; Baldoma, Laura; Aguilar, Juan

    2008-05-01

    When exposed to enteric pathogens intestinal epithelial cells produce several cytokines and other proinflammatory mediators. To date there is no evidence that the ether-lipid platelet-activating factor (PAF) is one of these mediators. Our results revealed a significant increase in PAF production by human colonic tissue 4 h after infection by enterohemorrhagic Escherichia coli (EHEC) or Salmonella enteritidis. PAF is produced in the gut by cells of the immune system in response to bacterial infection. To determine whether the epithelial cells of colonic mucosa might also modulate PAF levels, we carried out PAF quantification and analysis of the enzymes involved in PAF synthesis in 5-day-old (undifferentiated) or 28-day-old (differentiated) Caco-2 cell cultures. Infection of undifferentiated Caco-2 cells with either bacterium had no effect on PAF levels, whereas in differentiated cells, infection by S. enteritidis increased PAF levels. Following infection by S. enteritidis, there were no changes in the activity of dithiothreitol-insensitive choline phosphotransferase. However, the enzymes of the remodeling pathway cytosolic phospholipase A(2), which catalyzes the formation of the PAF precursor lysoPAF, and lysoPAF acetyltransferase, are activated in the infected epithelial cells. This response is Ca(2+)-dependent.

  18. Noni (Morinda citrifolia L.) Fruit Extracts Improve Colon Microflora and Exert Anti-Inflammatory Activities in Caco-2 Cells.

    Science.gov (United States)

    Huang, Hsin-Lun; Liu, Cheng-Tzu; Chou, Ming-Chih; Ko, Chien-Hui; Wang, Chin-Kun

    2015-06-01

    Intestinal microflora and inflammation are associated with the risk of inflammatory bowel diseases. Noni (Morinda citrifolia L.) has various bioactivities, but its effect on colon health remains unknown. This study focused on the effects of fermented noni fruit extracts on colon microflora and inflammation of colon epithelial cells. The anti-inflammatory activities of ethanol and ethyl acetate extracts on Caco-2 cells were evaluated including interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2). The growth of Lactobacillus and Bifidobacterium species was promoted by ethanol extract. Ethyl acetate extract decreased intracellular reactive oxygen species and significantly suppressed COX-2, IL-8, and prostaglandin E2 production and neutrophil chemotaxis by suppressing the translocation of the p65 subunit. Quercetin was the main contributor to the anti-inflammatory activity. The fermented noni fruit promoted probiotic growths and downregulated the intracellular oxidation and inflammation in Caco-2 cells. These results suggest that fermented noni fruit might protect against inflammatory diseases of the colon.

  19. Transport of chlorpromazine in the Caco-2 cell permeability assay: a kinetic study

    NARCIS (Netherlands)

    Broeders, J.J.W.; Eijkeren, J.C.H.; Blaauboer, B.J.; Hermens, J.L.M.

    2012-01-01

    The intestinal transport of compounds can be measured in vitro with Caco-2 cell monolayers. We took a closer look at the exposure and fate of a chemical in the Caco-2 cell assay, including the effect of protein binding. Transport of chlorpromazine (CPZ) was measured in the absorptive and secretory

  20. The mycotoxin patulin, modulates tight junctions in caco-2 cells.

    Science.gov (United States)

    McLaughlin, John; Lambert, Daniel; Padfield, Philip J; Burt, Julian P H; O'Neill, Catherine A

    2009-02-01

    The mycotoxin patulin is a common contaminant of fruit. Here, we demonstrate that patulin reduces the barrier properties of the intestinal cell line, caco-2 by specific effects on tight junction components. Within 5h of exposure to 100 microM toxin, the transepithelial electrical resistance of caco-2 monolayers was reduced by approximately 95% and the monolayer became more permeable to FITC-labelled dextrans of 4-40 kDa. Immunoblotting revealed occludin proteolysis and a significant reduction in ZO-1 levels. Patulin had no influence on claudin levels but marked changes in their distribution were observed. These data indicate that patulin decreases the barrier properties of caco-2 monolayers by modulation of the tight junction.

  1. Physiological levels of tea catechins increase cellular lipid antioxidant activity of vitamin C and vitamin E in human intestinal Caco-2 cells

    Science.gov (United States)

    Intra, Janjira; Kuo, Shiu-Ming

    2007-01-01

    Oxidative stress has been linked to the development of various chronic diseases. Vegetables and fruits, which contain polyphenols, were shown to have protective effects. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenol abundant in tea, has been shown to have antioxidant activities in cell-free conditions and this study focused on the effect of cellular EGCG. Using an intestinal cell model to examine the oxidative stress induced by hydroxyl radicals, we report here that physiological concentrations (0.1-1 μM) of EGCG have dose- and incubation duration-dependent cell-associated lipid antioxidant activity (measuring malondialdehyde production). Vitamin E and vitamin C at 10-40 μM also showed cell-associated lipid antioxidant activities under shorter incubation durations. When EGCG was included in the incubation with vitamin E or C, more antioxidant activities were consistently observed than when vitamins were added alone. Catechin (widely present in fruits and vegetables) at 1 μM also significantly increased the antioxidant activity of vitamin E and C. Previous studies examining cell-associated activity of EGCG mainly focused on the 10-100 μM concentration range. Our results suggest that although the physiological level (0.1-1 μM) of dietary catechins is much lower than that of vitamins, they further contribute to the total antioxidant capacity even in the presence of vitamins. PMID:17603031

  2. Disruption of Escherichia coli Nissle 1917 K5 capsule biosynthesis, through loss of distinct kfi genes, modulates interaction with intestinal epithelial cells and impact on cell health.

    Directory of Open Access Journals (Sweden)

    Jonathan Nzakizwanayo

    Full Text Available Escherichia coli Nissle 1917 (EcN is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants, prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB, or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC, were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.

  3. Omeprazole decreases magnesium transport across Caco-2 monolayers

    Science.gov (United States)

    Thongon, Narongrit; Krishnamra, Nateetip

    2011-01-01

    AIM: To elucidate the effect and underlying mechanisms of omeprazole action on Mg2+ transport across the intestinal epithelium. METHODS: Caco-2 monolayers were cultured in various dose omeprazole-containing media for 14 or 21 d before being inserted into a modified Ussing chamber apparatus to investigate the bi-directional Mg2+ transport and electrical parameters. Paracellular permeability of the monolayer was also observed by the dilution potential technique and a cation permeability study. An Arrhenius plot was performed to elucidate the activation energy of passive Mg2+ transport across the Caco-2 monolayers. RESULTS: Both apical to basolateral and basolateral to apical passive Mg2+ fluxes of omeprazole-treated epithelium were decreased in a dose- and time-dependent manner. Omeprazole also decreased the paracellular cation selectivity and changed the paracellular selective permeability profile of Caco-2 epithelium to Li+, Na+, K+, Rb+, and Cs+ from series VII to series VI of the Eisenman sequence. The Arrhenius plot revealed the higher activation energy for passive Mg2+ transport in omeprazole-treated epithelium than that of control epithelium, indicating that omeprazole affected the paracellular channel of Caco-2 epithelium in such a way that Mg2+ movement was impeded. CONCLUSION: Omeprazole decreased paracellular cation permeability and increased the activation energy for passive Mg2+ transport of Caco-2 monolayers that led to the suppression of passive Mg2+ absorption. PMID:21472124

  4. Effects of Supernatants from Escherichia coli Nissle 1917 and Faecalibacterium prausnitzii on Intestinal Epithelial Cells and a Rat Model of 5-Fluorouracil-Induced Mucositis.

    Science.gov (United States)

    Wang, Hanru; Jatmiko, Yoga D; Bastian, Susan E P; Mashtoub, Suzanne; Howarth, Gordon S

    2017-01-01

    Faecalibacterium prausnitzii (Fp) and Escherichia coli Nissle 1917 (EcN) are probiotics, which have been reported to ameliorate certain gastrointestinal disorders. We evaluated the effects of supernatants (SN) derived from Fp and EcN on 5-fluorouracil (5-FU)-treated intestinal cells and in a rat model of mucositis. In vitro: IEC-6, Caco-2, and T-84 cells were analyzed for viability and monolayer permeability. In vivo: Female dark agouti rats were gavaged with Fp or EcN SN and injected intraperitoneally with saline (control) or 5-FU to induce mucositis. Rats were euthanized and intestinal tissues collected for myeloperoxidase assay and histological analyses. In vitro: Caco-2 cell viability was further reduced when treated with Fp SN + 5-FU compared to 5-FU controls. In both Caco-2 and T-84 cells, Fp SN partially prevented the decrease in transepithelial electrical resistance (TER) caused by 5-FU administration. In vivo: 5-FU-injected rats administered Fp SN or EcN SN partly prevented body weight loss and normalized water intake compared to 5-FU controls. These results suggest a growth inhibitory mechanism of Fp SN action on transformed epithelial cells that could be mediated by effects on tight junctions. Factors derived from Fp SN and EcN SN could have a role in reducing the severity of intestinal mucositis.

  5. Lactobacillus acidophilus counteracts enteropathogenic E. coli-induced inhibition of butyrate uptake in intestinal epithelial cells.

    Science.gov (United States)

    Kumar, Anoop; Alrefai, Waddah A; Borthakur, Alip; Dudeja, Pradeep K

    2015-10-01

    Butyrate, a key short-chain fatty acid metabolite of colonic luminal bacterial action on dietary fiber, serves as a primary fuel for the colonocytes, ameliorates mucosal inflammation, and stimulates NaCl absorption. Absorption of butyrate into the colonocytes is essential for these intracellular effects. Monocarboxylate transporter 1 (MCT1) plays a major role in colonic luminal butyrate absorption. Previous studies (Tan J, McKenzie C, Potamitis M, Thorburn AN, Mackay CR, Macia L. Adv Immunol 121: 91-119, 2014.) showed decreased MCT1 expression and function in intestinal inflammation. We have previously shown (Borthakur A, Gill RK, Hodges K, Ramaswamy K, Hecht G, Dudeja PK. Am J Physiol Gastrointest Liver Physiol 290: G30-G35, 2006.) impaired butyrate absorption in human intestinal epithelial Caco-2 cells due to decreased MCT1 level at the apical cell surface following enteropathogenic E. coli (EPEC) infection. Current studies, therefore, examined the potential role of probiotic Lactobacilli in stimulating MCT1-mediated butyrate uptake and counteracting EPEC inhibition of MCT1 function. Of the five species of Lactobacilli, short-term (3 h) treatment with L. acidophilus (LA) significantly increased MCT1-mediated butyrate uptake in Caco-2 cells. Heat-killed LA was ineffective, whereas the conditioned culture supernatant of LA (LA-CS) was equally effective in stimulating MCT1 function, indicating that the effects are mediated by LA-secreted soluble factor(s). Furthermore, LA-CS increased apical membrane levels of MCT1 protein via decreasing its basal endocytosis, suggesting that LA-CS stimulation of butyrate uptake could be secondary to increased levels of MCT1 on the apical cell surface. LA-CS also attenuated EPEC inhibition of butyrate uptake and EPEC-mediated endocytosis of MCT1. Our studies highlight distinct role of specific LA-secreted molecules in modulating colonic butyrate absorption.

  6. Krill oil reduces intestinal inflammation by improving epithelial integrity and impairing adherent-invasive Escherichia coli pathogenicity.

    Science.gov (United States)

    Costanzo, Manuela; Cesi, Vincenzo; Prete, Enrica; Negroni, Anna; Palone, Francesca; Cucchiara, Salvatore; Oliva, Salvatore; Leter, Beatrice; Stronati, Laura

    2016-01-01

    Krill oil is a marine derived oil rich in phospholipids, astaxanthin and omega-3 fatty acids. Several studies have found benefits of krill oil against oxidative and inflammatory damage. We aimed at assessing the ability of krill oil to reduce intestinal inflammation by improving epithelial barrier integrity, increasing cell survival and reducing pathogenicity of adherent-invasive Escherichia coli. CACO2 and HT29 cells were exposed to cytomix (TNFα and IFNγ) to induce inflammation and co-exposed to cytomix and krill oil. E-cadherin, ZO-1 and F-actin levels were analyzed by immunofluorescence to assess barrier integrity. Scratch test was performed to measure wound healing. Cell survival was analyzed by flow cytometry. Adherent-invasive Escherichia coli LF82 was used for adhesion/invasion assay. In inflamed cells E-cadherin and ZO-1 decreased, with loss of cell-cell adhesion, and F-actin polymerization increased stress fibres; krill oil restored initial conditions and improved wound healing, reduced bacterial adhesion/invasion in epithelial cells and survival within macrophages; krill oil reduced LF82-induced mRNA expression of pro-inflammatory cytokines. Krill oil improves intestinal barrier integrity and epithelial restitution during inflammation and controls bacterial adhesion and invasion to epithelial cells. Thus, krill oil may represent an innovative tool to reduce intestinal inflammation. Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  7. SMS regulates the expression and function of P-gp and MRP2 in Caco-2 cells.

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    Jin, Guiying; Li, Yang; Zhu, Yuwen; Du, Lisha; Yan, Junkai; Yang, Qing

    2016-12-01

    Sphingomyelin synthase (SMS) has two isoforms of SMS1 and SMS2, the last enzyme involved in the biosynthesis of sphingomyelin (SM), and has impact on the expression of membrane proteins. In the present study, we explored the potential effects of SMS on drug transporters, a special family of membrane proteins in human intestinal epithelial Caco-2 cells. The specific knockdown of SMS1 or SMS2 with siRNA in Caco-2 cells substantially decreased the expression and function of P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2) rather than other drug transporters MRP1, MRP3, PEPT1, OATP2B1, and BCRP. In the SMS1 stable overexpressed Caco-2 cell line, the expression levels of P-gp and MRP2 and transcription factor pregnane X receptor (PXR) were upregulated and the phosphorylation levels of signaling pathways janus protein tyrosine kinase 2 (JAK-2) and extracellular signal-regulated kinases (ERK) were also evidently increased; however, the upregulated mRNA expression levels of PXR, P-gp, and MRP2 were diminished by inhibiting the phosphorylation of ERK and JAK-2. Furthermore, the SMS1 overexpression in Caco-2 cells altered the expression levels of ERM proteins ezrin and moesin, which are closely connected to the function of drug transporters. In conclusion, we herein demonstrate for the first time that in Caco-2 cells SMS regulates the expression and function of drug transporters P-gp and MRP2, and their regulator PXR is mediated by phosphorylated ERK and JAK-2 signaling pathways.

  8. Cyanidin-3-O-Glucoside Modulates the In Vitro Inflammatory Crosstalk between Intestinal Epithelial and Endothelial Cells

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    Daniela Ferrari

    2017-01-01

    Full Text Available Intestinal epithelium represents a protective physical barrier and actively contributes to the mucosal immune system. Polarized basolateral intestinal secretion of inflammatory mediators, followed by activation of NF-κB signaling and inflammatory pathways in endothelial cells, efficiently triggers extravasation of neutrophils from the vasculature, therefore contributing to the development and maintenance of intestinal inflammation. Proper regulation of NF-κB activation at the epithelial interface is crucial for the maintenance of physiological tissue homeostasis. Many papers reported that anthocyanins, a group of compounds belonging to flavonoids, possess anti-inflammatory effects and modulate NF-κB activity. In this study, by using a coculture in vitro system, we aimed to evaluate the effects of TNF-α-stimulated intestinal cells on endothelial cells activation, as well as the protective effects of cyanidin-3-glucoside (C3G. In this model, TNF-α induced nuclear translocation of NF-κB and TNF-α and IL-8 gene expression in Caco-2 cells, whereas C3G pretreatment dose-dependently reduced these effects. Furthermore, TNF-α-stimulated Caco-2 cells induced endothelial cells activation with increased E-selectin and VCAM-1 mRNA, leukocyte adhesion, and NF-κB levels in HUVECs, which were inhibited by C3G. We demonstrated that selective inhibition of the NF-κB pathway in epithelial cells represents the main mechanism by which C3G exerts these protective effects. Thus, anthocyanins could contribute to the management of chronic gut inflammatory diseases.

  9. Boswellia serrata Preserves Intestinal Epithelial Barrier from Oxidative and Inflammatory Damage.

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    Daniela Catanzaro

    Full Text Available Aminosalicylates, corticosteroids and immunosuppressants are currently the therapeutic choices in inflammatory bowel diseases (IBD, however, with limited remission and often serious side effects. Meanwhile complementary and alternative medicine (CAM use is increasing, particularly herbal medicine. Boswellia serrata is a traditional Ayurvedic remedy with anti-inflammatory properties, of interest for its usefulness in IBDs. The mechanism of this pharmacological potential of Boswellia serrata was investigated in colonic epithelial cell monolayers exposed to H2O2 or INF-γ+TNF-α, chosen as in vitro experimental model of intestinal inflammation. The barrier function was evaluated by the transepithelial electrical resistance (TEER and paracellular permeability assay, and by the tight junction proteins (zonula occludens-1, ZO-1 and occludin immunofluorescence. The expression of phosphorylated NF-κB and reactive oxygen species (ROS generation were determined by immunoblot and cytofluorimetric assay, respectively. Boswellia serrata oleo-gum extract (BSE and its pure derivative acetyl-11-keto-β-boswellic acid (AKBA, were tested at 0.1-10 μg/ml and 0.027 μg/ml, respectively. BSE and AKBA safety was demonstrated by no alteration of intestinal cell viability and barrier function and integrity biomarkers. H2O2 or INF-γ+TNF-α treatment of Caco-2 cell monolayers significantly reduced TEER, increased paracellular permeability and caused the disassembly of tight junction proteins occludin and ZO-1. BSE and AKBA pretreatment significantly prevented functional and morphological alterations and also the NF-κB phosphorylation induced by the inflammatory stimuli. At the same concentrations BSE and AKBA counteracted the increase of ROS caused by H2O2 exposure. Data showed the positive correlation of the antioxidant activity with the mechanism involved in the physiologic maintenance of the integrity and function of the intestinal epithelium. This study

  10. DNA-fragments are transcytosed across CaCo-2 cells by adsorptive endocytosis and vesicular mediated transport.

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    Lene E Johannessen

    Full Text Available Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB. After 90 min the difference in directionality AB vs. BA was >10(3 fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system.

  11. Influenza A virus infection of intestinal epithelial cells enhances the adhesion ability of Crohn's disease associated Escherichia coli strains.

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    Aleandri, Marta; Conte, Maria Pia; Simonetti, Giovanna; Panella, Simona; Celestino, Ignacio; Checconi, Paola; Marazzato, Massimiliano; Longhi, Catia; Goldoni, Paola; Nicoletti, Mauro; Barnich, Nicolas; Palamara, Anna Teresa; Schippa, Serena; Nencioni, Lucia

    2015-01-01

    Modifications of intestinal glycoreceptors expression, in particular CEACAM6, typically found in ileal Crohn's disease (CD), favor, among the commensal species of microbiota, the enrichment in Escherichia coli. Removal of protein glycosidic residues by neuraminidase, a sialidase typical of influenza virus, increases adhesion ability of Escherichia coli to Caco-2 intestinal cells. In this study we investigated whether influenza virus infection of human intestinal epithelial cells could influence the adhesiveness of different Escherichia coli strains isolated from CD patients by altering surface glycoreceptors. Influenza virus infection of intestinal cells increased exposure of galactose and mannose residues on the cell surface. In particular, glycoreceptors Thomsen-Friedenreich and CEACAM6 were over-expressed in influenza virus infected cells. In the same experimental conditions, a significant increase in bacterial adhesiveness was observed, independently of their own adhesive ability. The increase was reverted by treatment with anti-TF and anti-CEACAM6 antibodies. Interestingly, influenza virus was able to efficiently replicate in human primary intestinal cells leading to TF exposure. Finally, intestinal infected cells produced high levels of pro-inflammatory cytokines compared to control. Overall these data suggest that influenza virus infection, could constitute an additional risk factor in CD patients.

  12. The impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation.

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    Majka, Grzegorz; Więcek, Grażyna; Śróttek, Małgorzata; Śpiewak, Klaudyna; Brindell, Małgorzata; Koziel, Joanna; Marcinkiewicz, Janusz; Strus, Magdalena

    2016-12-01

    Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host's health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis.

  13. A Metronidazole-Resistant Isolate of Blastocystis spp. Is Susceptible to Nitric Oxide and Downregulates Intestinal Epithelial Inducible Nitric Oxide Synthase by a Novel Parasite Survival Mechanism ▿

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    Mirza, Haris; Wu, Zhaona; Kidwai, Fahad; Tan, Kevin S. W.

    2011-01-01

    Blastocystis, one of the most common parasites colonizing the human intestine, is an extracellular, noninvasive, luminal protozoan with controversial pathogenesis. Blastocystis infections can be asymptomatic or cause intestinal symptoms of vomiting, diarrhea, and abdominal pain. Although chronic infections are frequently reported, Blastocystis infections have also been reported to be self-limiting in immunocompetent patients. Characterizing the host innate response to Blastocystis would lead to a better understanding of the parasite's pathogenesis. Intestinal epithelial cells produce nitric oxide (NO), primarily on the apical side, in order to target luminal pathogens. In this study, we show that NO production by intestinal cells may be a host defense mechanism against Blastocystis. Two clinically relevant isolates of Blastocystis, ST-7 (B) and ST-4 (WR-1), were found to be susceptible to a range of NO donors. ST-7 (B), a metronidazole-resistant isolate, was found to be more sensitive to nitrosative stress. Using the Caco-2 model of human intestinal epithelium, Blastocystis ST-7 (B) but not ST-4 (WR-1) exhibited dose-dependent inhibition of Caco-2 NO production, and this was associated with downregulation of inducible nitric oxide synthase (iNOS). Despite its higher susceptibility to NO, Blastocystis ST-7 (B) may have evolved unique strategies to evade this potential host defense by depressing host NO production. This is the first study to highlight a strain-to-strain variation in the ability of Blastocystis to evade the host antiparasitic NO response. PMID:21930763

  14. A metronidazole-resistant isolate of Blastocystis spp. is susceptible to nitric oxide and downregulates intestinal epithelial inducible nitric oxide synthase by a novel parasite survival mechanism.

    Science.gov (United States)

    Mirza, Haris; Wu, Zhaona; Kidwai, Fahad; Tan, Kevin S W

    2011-12-01

    Blastocystis, one of the most common parasites colonizing the human intestine, is an extracellular, noninvasive, luminal protozoan with controversial pathogenesis. Blastocystis infections can be asymptomatic or cause intestinal symptoms of vomiting, diarrhea, and abdominal pain. Although chronic infections are frequently reported, Blastocystis infections have also been reported to be self-limiting in immunocompetent patients. Characterizing the host innate response to Blastocystis would lead to a better understanding of the parasite's pathogenesis. Intestinal epithelial cells produce nitric oxide (NO), primarily on the apical side, in order to target luminal pathogens. In this study, we show that NO production by intestinal cells may be a host defense mechanism against Blastocystis. Two clinically relevant isolates of Blastocystis, ST-7 (B) and ST-4 (WR-1), were found to be susceptible to a range of NO donors. ST-7 (B), a metronidazole-resistant isolate, was found to be more sensitive to nitrosative stress. Using the Caco-2 model of human intestinal epithelium, Blastocystis ST-7 (B) but not ST-4 (WR-1) exhibited dose-dependent inhibition of Caco-2 NO production, and this was associated with downregulation of inducible nitric oxide synthase (iNOS). Despite its higher susceptibility to NO, Blastocystis ST-7 (B) may have evolved unique strategies to evade this potential host defense by depressing host NO production. This is the first study to highlight a strain-to-strain variation in the ability of Blastocystis to evade the host antiparasitic NO response.

  15. Proposing a Caco-2/HepG2 cell model for in vitro iron absorption studies.

    Science.gov (United States)

    Scheers, Nathalie M; Almgren, Annette B; Sandberg, Ann-Sofie

    2014-07-01

    The Caco-2 cell line is well established as an in vitro model for iron absorption. However, the model does not reflect the regulation of iron absorption by hepcidin produced in the liver. We aimed to develop the Caco-2 model by introducing human liver cells (HepG2) to Caco-2 cells. The Caco-2 and HepG2 epithelia were separated by a liquid compartment, which allowed for epithelial interaction. Ferritin levels in cocultured Caco-2 controls were 21.7±10.3 ng/mg protein compared to 7.7±5.8 ng/mg protein in monocultured Caco-2 cells. The iron transport across Caco-2 layers was increased when liver cells were present (8.1%±1.5% compared to 3.5%±2.5% at 120 μM Fe). Caco-2 cells were exposed to 0, 80 and 120 μM Fe and responded with increased hepcidin production at 120 μM Fe (3.6±0.3 ng/ml compared to 2.7±0.3 ng/ml). The expression of iron exporter ferroportin in Caco-2 cells was decreased at the hepcidin concentration of 3.6 ng/ml and undetectable at external addition of hepcidin (10 ng/ml). The apical transporter DMT1 was also undetectable at 10 ng/ml but was unchanged at the lower concentrations. In addition, we observed that sourdough bread, in comparison to heat-treated bread, increased the bioavailability of iron despite similar iron content (53% increase in ferritin formation, 97% increase in hepcidin release). This effect was not observed in monocultured Caco-2 cells. The Caco-2/HepG2 model provides an alternative approach to in vitro iron absorption studies in which the hepatic regulation of iron transport must be considered. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Activation of epithelial STAT3 regulates intestinal homeostasis.

    Science.gov (United States)

    Neufert, Clemens; Pickert, Geethanjali; Zheng, Yan; Wittkopf, Nadine; Warntjen, Moritz; Nikolaev, Alexei; Ouyang, Wenjun; Neurath, Markus F; Becker, Christoph

    2010-02-15

    The intestinal epithelium that lines the mucosal surface along the GI-tract is a key player for the intestinal homeostasis of the healthy individual. In case of a mucosal damage or a barrier defect as seen in patients with inflammatory bowel disease, the balance is disturbed, and translocation of intestinal microbes to the submucosa is facilitated. We recently demonstrated a pivotal role of STAT3 activation in intestinal epithelial cells (IEC) for the restoration of the balance at the mucosal surface of the gut in an experimental colitis model. STAT3 was rapidly induced in intestinal epithelial cells upon challenge of mice in both experimental colitis and intestinal wound healing models. STAT3 activation was found to be dispensable in the steady-state conditions but was important for efficient regeneration of the epithelium in response to injury. Here, we extend our previous findings by showing epithelial STAT3 activation in human patients suffering from IBD and provide additional insights how the activation of epithelial STAT3 by IL-22 regulates intestinal homeostasis and mucosal wound healing. We also demonstrate that antibody-mediated neutralization of IL-22 has little impact on the development of experimental colitis in mice, but significantly delays recovery from colitis. Thus, our data suggest that targeting the STAT3 signaling pathway in IEC is a promising therapeutic approach in situations when the intestinal homeostasis is disturbed, e.g., as seen in Crohn's disease or Ulcerative colitis.

  17. Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells.

    Science.gov (United States)

    Zeitouni, Nathalie E; Dersch, Petra; Naim, Hassan Y; von Köckritz-Blickwede, Maren

    2016-01-01

    Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β1 integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β1 integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β1 integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β1 integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β1 integrins.

  18. The P2Y6 receptor mediates Clostridium difficile toxin-induced CXCL8/IL-8 production and intestinal epithelial barrier dysfunction.

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    Ashleigh Hansen

    Full Text Available C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI is driven by toxin A (TcdA and toxin B (TcdB, secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y6 receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y6 inhibitor (MRS2578. This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y6 receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y6 receptor antagonists (MSR2578 attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y6 receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.

  19. Effects of cadmium chloride on the paracellular barrier function of intestinal epithelial cell lines

    NARCIS (Netherlands)

    Duizer, E.; Gilde, A.J.; Versantvoort, C.H.M.; Groten, J.P.

    1999-01-01

    In the present study we characterized the functional and structural disruption of the paracellular barrier of intestinal epithelium in vitro in relation to cytotoxicity after apical Cd2+ exposure. For that purpose filter-grown Caco-2 and IEC-18 cells were apically exposed to 5 to 100 μM CdCl2 for 4

  20. Mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge.

    Science.gov (United States)

    Bannunah, Azzah M; Vllasaliu, Driton; Lord, Jennie; Stolnik, Snjezana

    2014-12-01

    This study investigated the effect of nanoparticle size (50 and 100 nm) and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number, and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below half maximal effective concentration (EC₅₀) revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride), and under cholesterol depletion (38%; via methyl-β-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in

  1. Simultaneous assessment of absorption characteristics of coumarins from Angelicae Pubescentis Radix: In vitro transport across Caco-2 cell and in vivo pharmacokinetics in rats after oral administration.

    Science.gov (United States)

    Yang, Yan-Fang; Zhang, Lei; Zhang, You-Bo; Yang, Xiu-Wei

    2017-08-15

    Angelicae Pubescentis Radix (APR), a well-known traditional Chinese medicine, is widely used for the treatments of rheumatism and headache for centuries. To assess the absorption characteristics of coumarins from APR, a sensitive and reliable UPLC-MS/MS method was established for the simultaneous determination of sixteen coumarins from APR, including psoralen, xanthotoxin, bergapten, bergaptol, isoimperatorin, imperatorin, columbianetin, columbianetin acetate, columbianadin, oxypeucedanin hydrate, angelol B, umbelliferone, scopoletin, osthole, meranzin hydrate and nodakenetin. The specificity, linearity, sensitivity, precision, accuracy, recovery, matrix effect and stability of the method were all validated to be satisfactory. The method was then applied to the in vitro transport of APR extract (APRE) across human intestinal epithelial Caco-2 cell and in vivo pharmacokinetics in rats after oral administration of APRE. All of the tested coumarins were well or moderately absorbed across Caco-2 monolayers, and could be quickly absorbed into rat blood circulation after oral administration. Columbianetin was the most easily absorbed compound across Caco-2 cell, and also had extremely highest plasma concentration in vivo. Excellent correlation between in vitro absorption across Caco-2 cell monolayers and in vivo pharmacokinetics of coumarins from APRE was well verified. The results provided valuable information for the overall absorption characteristics of the coumarins from APR, as well as for its further studies of in vivo active substances in the further. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Caco-2 Cells, Biopharmaceutics Classification System (BCS and Biowaiter

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    Libuše Smetanová

    2011-01-01

    Full Text Available Almost all orally administered drugs are absorbed across the intestinal mucosa. The Caco-2 monolayers are used as an in vitro model to predict drug absorption in humans and to explore mechanism of drug absorption. The Caco-2 cells are derived from a human colon adenocarcinoma and spontaneously differentiate to form confluent monolayer of polarized cells structurally and functionally resembling the small intestinal epithelium. For studying drug permeability, Caco-2 cells are seeded onto the Transwell inserts with semipermeable membrane and grown to late confluence (21 days. After determination of cell viability, the integrity of monolayer is checked by phenol red permeability and by 14C-mannitol permeability. The transport from apical to basolateral (AP-BL and basolateral to apical (BL-AP is studied by adding the diluted drug on the apical or basolateral side and withdrawing the samples from the opposite compartment, respectively, for HPLC analysis or liquid scintillation spectrometry. Ca2+- free transport medium is used to determine paracellular component of the drug transport. On the basis of permeability and solubility, drugs can be categorized into four classes of Biopharmaceutics Classification System (BCS. For certain drugs, the BCS-based biowaiver approach can be used which enables to reduce in vivo bioequivalence studies.

  3. Epithelial NEMO links innate immunity to chronic intestinal inflammation.

    Science.gov (United States)

    Nenci, Arianna; Becker, Christoph; Wullaert, Andy; Gareus, Ralph; van Loo, Geert; Danese, Silvio; Huth, Marion; Nikolaev, Alexei; Neufert, Clemens; Madison, Blair; Gumucio, Deborah; Neurath, Markus F; Pasparakis, Manolis

    2007-03-29

    Deregulation of intestinal immune responses seems to have a principal function in the pathogenesis of inflammatory bowel disease. The gut epithelium is critically involved in the maintenance of intestinal immune homeostasis-acting as a physical barrier separating luminal bacteria and immune cells, and also expressing antimicrobial peptides. However, the molecular mechanisms that control this function of gut epithelial cells are poorly understood. Here we show that the transcription factor NF-kappaB, a master regulator of pro-inflammatory responses, functions in gut epithelial cells to control epithelial integrity and the interaction between the mucosal immune system and gut microflora. Intestinal epithelial-cell-specific inhibition of NF-kappaB through conditional ablation of NEMO (also called IkappaB kinase-gamma (IKKgamma)) or both IKK1 (IKKalpha) and IKK2 (IKKbeta)-IKK subunits essential for NF-kappaB activation-spontaneously caused severe chronic intestinal inflammation in mice. NF-kappaB deficiency led to apoptosis of colonic epithelial cells, impaired expression of antimicrobial peptides and translocation of bacteria into the mucosa. Concurrently, this epithelial defect triggered a chronic inflammatory response in the colon, initially dominated by innate immune cells but later also involving T lymphocytes. Deficiency of the gene encoding the adaptor protein MyD88 prevented the development of intestinal inflammation, demonstrating that Toll-like receptor activation by intestinal bacteria is essential for disease pathogenesis in this mouse model. Furthermore, NEMO deficiency sensitized epithelial cells to tumour-necrosis factor (TNF)-induced apoptosis, whereas TNF receptor-1 inactivation inhibited intestinal inflammation, demonstrating that TNF receptor-1 signalling is crucial for disease induction. These findings demonstrate that a primary NF-kappaB signalling defect in intestinal epithelial cells disrupts immune homeostasis in the gastrointestinal tract

  4. Expression of Lactobacillus reuteri Pg4 collagen-binding protein gene in Lactobacillus casei ATCC 393 increases its adhesion ability to Caco-2 cells.

    Science.gov (United States)

    Hsueh, Hsiang-Yun; Yueh, Pei-Ying; Yu, Bi; Zhao, Xin; Liu, Je-Ruei

    2010-12-08

    The collagen-binding protein gene cnb was cloned from the probiotic Lactobacillus reuteri strain Pg4. The DNA sequence of the cnb gene (792 bp) has an open reading frame encoding 263 amino acids with a calculated molecular weight of 28.5 kDa. The cnb gene was constructed so as to constitutively express under the control of the Lactococcus lactis lacA promoter and was transformed into Lactobacillus casei ATCC 393, a strain isolated from dairy products with poor ability to adhere to intestinal epithelial cells. Confocal immunofluorescence microscopic and flow cytometric analysis of the transformed strain Lb. casei pNZ-cnb indicated that Cnb was displayed on its cell surface. Lb. casei pNZ-cnb not only showed a higher ability to adhere to Caco-2 cells but also exhibited a higher competition ability against Escherichia coli O157:H7 and Listeria monocytogenes adhesion to Caco-2 cells than Lb. casei ATCC 393.

  5. Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing

    Science.gov (United States)

    Using the small intestine enterocyte Caco-2 cell model, sucrase-isomaltase (SI, the mucosal alpha-glucosidase complex) expression and modification were examined relative to exposure to different mono- and disaccharide glycemic carbohydrates. Caco-2/TC7 cells were grown on porous supports to post-con...

  6. Iron availability increases the pathogenic potential of Salmonella typhimurium and other enteric pathogens at the intestinal epithelial interface.

    Directory of Open Access Journals (Sweden)

    Guus A M Kortman

    Full Text Available Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration (P = 0.0001, whereas this was not the case for the non-pathogenic Lactobacillus plantarum (P = 0.42. Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.

  7. Effects of ethanol and acetaldehyde on tight junction integrity: in vitro study in a three dimensional intestinal epithelial cell culture model.

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    Elhaseen Elamin

    Full Text Available BACKGROUND: Intestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the blood after moderate ethanol consumption, of its metabolite acetaldehyde and of the combination of both compounds on intestinal barrier function in a three-dimensional cell culture model. METHODS AND FINDINGS: Caco-2 cells were grown in a basement membrane matrix (Matrigel™ to induce spheroid formation and were then exposed to the compounds at the basolateral side. Morphological differentiation of the spheroids was assessed by immunocytochemistry and transmission electron microscopy. The barrier function was assessed by the flux of FITC-labeled dextran from the basal side into the spheroids' luminal compartment using confocal microscopy. Caco-2 cells grown on Matrigel assembled into fully differentiated and polarized spheroids with a central lumen, closely resembling enterocytes in vivo and provide an excellent model to study epithelial barrier functionality. Exposure to ethanol (10-40 mM or acetaldehyde (25-200 µM for 3 h, dose-dependently and additively increased the paracellular permeability and induced redistribution of ZO-1 and occludin without affecting cell viability or tight junction-encoding gene expression. Furthermore, ethanol and acetaldehyde induced lysine residue and microtubules hyperacetylation. CONCLUSIONS: These results indicate that ethanol at concentrations found in the blood after moderate drinking and acetaldehyde, alone and in combination, can increase the intestinal epithelial permeability. The data also point to the involvement of protein hyperacetylation in

  8. Primary mouse small intestinal epithelial cell cultures

    NARCIS (Netherlands)

    Sato, T.; Clevers, H.

    2013-01-01

    The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently shown that Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) is expressed in intestinal stem cells by an in vivo genetic lineage tracing strategy. In the past, extensive efforts have

  9. Chrysosplenetin inhibits artemisinin efflux in P-gp-over-expressing Caco-2 cells and reverses P-gp/MDR1 mRNA up-regulated expression induced by artemisinin in mouse small intestine.

    Science.gov (United States)

    Ma, Liping; Wei, Shijie; Yang, Bei; Ma, Wei; Wu, Xiuli; Ji, Hongyan; Sui, Hong; Chen, Jing

    2017-12-01

    CYP3A4 and P-gp together form a highly efficient barrier for orally absorbed drugs and always share the same substrates. Our previous work revealed that chrysosplenetin (CHR) significantly augmented the rat plasma level and anti-malarial efficacy of artemisinin (ART), partially due to the uncompetitive inhibition effect of CHR on rat CYP3A. But the impact of CHR on P-gp is still unknown. The present study investigates whether CHR interferes with P-gp-mediated efflux of ART and elucidates the underlying mechanism. P-gp-over-expressing Caco-2 cells were treated with ART (10 μM) or ART-CHR (1:2, 10:20 μM) in ART bidirectional transport experiment. ART concentration was determined by UHPLC-MS/MS method. Healthy male ICR mice were randomly divided into nine groups (n = 6) including negative control (0.5% CMC-Na solution, 13 mL/kg), ART alone (40 mg/kg), verapamil (positive control, 40 mg/kg), ART-verapamil (1:1, 40:40 mg/kg), CHR alone (80 mg/kg), ART-CHR (1:0.1, 40:4 mg/kg), ART-CHR (1:1, 40:40 mg/kg), ART-CHR (1:2, 40:80 mg/kg) and ART-CHR (1:4, 40:160 mg/kg). The drugs were administrated intragastrically for seven consecutive days. MDR1 and P-gp expression levels in mice small intestine were examined by performing RT-PCR and western blot analysis. ABC coupling ATPase activity was also determined. After combined with CHR (1:2), Papp (AP-BL) and Papp (BL-AP) of ART changed to 4.29 × 10 (-) (8) (increased 1.79-fold) and 2.85 × 10 (-) (8 )cm/s (decreased 1.24-fold) from 2.40 × 10 (-) (8) and 3.54 × 10 (-) (8 )cm/s, respectively. Efflux ratio (PBA/PAB) declined 2.21-fold (p P-gp levels compared with vehicle, while CHR in combination ratio of 0:1, 0.1:1, 1:1, 2:1 and 4:1 with ART, reversed them to normal levels as well as negative control (p p P-gp activity and reverse the up-regulated P-gp and MDR1 levels induced by ART. It suggested that CHR potentially can be used as a P-gp reversal agent to

  10. Intestinal alkaline phosphatase contributes to the reduction of severe intestinal epithelial damage.

    NARCIS (Netherlands)

    Bol-Schoenmakers, M.|info:eu-repo/dai/nl/304846953; Fiechter, D.|info:eu-repo/dai/nl/331422018; Raaben, W.; Hassing, I.; Bleumink, R.; Kruijswijk, D.; Maijoor, K.; Tersteeg-Zijderveld, M.; Brands, R.; Pieters, R.|info:eu-repo/dai/nl/35112148X

    2010-01-01

    Inflammatory bowel disease is characterized by chronic inflammation of the intestine and is accompanied by damage of the epithelial lining and by undesired immune responses towards enteric bacteria. It has been demonstrated that intestinal alkaline phosphatase (iAP) protects against the induction of

  11. Lycopene Modulates THP1 and Caco2 Cells Inflammatory State through Transcriptional and Nontranscriptional Processes

    Science.gov (United States)

    Makon-Sébastien, Njock; Francis, Fouchier; Eric, Seree; Henri, Villard Pierre; François, Landrier Jean; Laurent, Pechere; Yves, Barra; Serge, Champion

    2014-01-01

    We revisited the action of a carotenoid, the lycopene, on the expression of proinflammatory genes, reactive oxygen species (ROS) production, and metalloprotease (MMP9) activity. THP1 and Caco2 cell lines were used as in vitro models for the two main cell types found in intestine tissue, that is, monocytes and epithelial cells. Proinflammatory condition was induced using either phorbol ester acetate (PMA), lipopolysaccharide (LPS) or tumor necrosis factor (TNF). In THP1 cells, short term pretreatment (2 h) with a low concentration (2 μM) of lycopene reinforce proinflammatory gene expression. The extent of the effect of lycopene is dependent on the proinflammtory stimulus (PMA, LPS or TNF) used. Lycopene enhanced MMP9 secretion via a c-AMP-dependent process, and reduced ROS production at higher concentrations than 2 μM. Cell culture media, conditioned by PMA-treated monocytes and then transferred on CaCo-2 epithelial cells, induced a proinflammatory state in these cells. The extent of this inflammatory effect was reduced when cells has been pretreated (12 h) with lycopene. At low concentration (2 μM or less), lycopene appeared to promote an inflammatory state not correlated with ROS modulation. At higher concentration (5 μM–20 μM), an anti-inflammatory effect takes place as a decrease of ROS production was detected. So, both concentration and time have to be considered in order to define the exact issue of the effect of carotenoids present in meals. PMID:24891766

  12. Lycopene Modulates THP1 and Caco2 Cells Inflammatory State through Transcriptional and Nontranscriptional Processes

    Directory of Open Access Journals (Sweden)

    Njock Makon-Sébastien

    2014-01-01

    Full Text Available We revisited the action of a carotenoid, the lycopene, on the expression of proinflammatory genes, reactive oxygen species (ROS production, and metalloprotease (MMP9 activity. THP1 and Caco2 cell lines were used as in vitro models for the two main cell types found in intestine tissue, that is, monocytes and epithelial cells. Proinflammatory condition was induced using either phorbol ester acetate (PMA, lipopolysaccharide (LPS or tumor necrosis factor (TNF. In THP1 cells, short term pretreatment (2 h with a low concentration (2 μM of lycopene reinforce proinflammatory gene expression. The extent of the effect of lycopene is dependent on the proinflammtory stimulus (PMA, LPS or TNF used. Lycopene enhanced MMP9 secretion via a c-AMP-dependent process, and reduced ROS production at higher concentrations than 2 μM. Cell culture media, conditioned by PMA-treated monocytes and then transferred on CaCo-2 epithelial cells, induced a proinflammatory state in these cells. The extent of this inflammatory effect was reduced when cells has been pretreated (12 h with lycopene. At low concentration (2 μM or less, lycopene appeared to promote an inflammatory state not correlated with ROS modulation. At higher concentration (5 μM–20 μM, an anti-inflammatory effect takes place as a decrease of ROS production was detected. So, both concentration and time have to be considered in order to define the exact issue of the effect of carotenoids present in meals.

  13. Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

    NARCIS (Netherlands)

    P. Aparicio-Domingo (Patricia); M. Romera-Hernandez (Monica); J.J. Karrich (Julien J.); F.H.J. Cornelissen (Ferry); N. Papazian (Natalie); D.J. Lindenbergh-Kortleve (Dicky); J.A. Butler (James A.); L. Boon (Louis); M. Coles (Mark); J.N. Samsom (Janneke); T. Cupedo (Tom)

    2015-01-01

    textabstractDisruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence

  14. New insights into mycotoxin mixtures: the toxicity of low doses of Type B trichothecenes on intestinal epithelial cells is synergistic.

    Science.gov (United States)

    Alassane-Kpembi, Imourana; Kolf-Clauw, Martine; Gauthier, Thierry; Abrami, Roberta; Abiola, François A; Oswald, Isabelle P; Puel, Olivier

    2013-10-01

    Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. DON is often present with other type B trichothecenes such as 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). Although the cytotoxicity of individual mycotoxins has been widely studied, data on the toxicity of mycotoxin mixtures are limited. The aim of this study was to assess interactions caused by co-exposure to Type B trichothecenes on intestinal epithelial cells. Proliferating Caco-2 cells were exposed to increasing doses of Type B trichothecenes, alone or in binary or ternary mixtures. The MTT test and neutral red uptake, respectively linked to mitochondrial and lysosomal functions, were used to measure intestinal epithelial cytotoxicity. The five tested mycotoxins had a dose-dependent effect on proliferating enterocytes and could be classified in increasing order of toxicity: 3-ADONmycotoxin combinations were synergistic; however DON-NIV-FX mixture showed antagonism. At higher concentrations (cytotoxic effect around 50%), the combinations had an additive or nearly additive effect. These results indicate that the simultaneous presence of low doses of mycotoxins in food commodities and diet may be more toxic than predicted from the mycotoxins alone. Considering the frequent co-occurrence of trichothecenes in the diet and the concentrations of toxins to which consumers are exposed, this synergy should be taken into account. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Impacts of Salmonella enterica Serovar Typhimurium and Its speG Gene on the Transcriptomes of In Vitro M Cells and Caco-2 Cells

    Science.gov (United States)

    Wang, Ke-Chuan; Huang, Chih-Hung; Huang, Ching-Jou

    2016-01-01

    Microfold or membranous (M) cells are specialized intestinal epithelial cells responsible for host immunity. The speG mutant of Salmonella Typhimurium (S. Typhimurium) is a nonreplicating strain within human cells to be a candidate vaccine vector for interacting with M cells. We conducted this study to identify the genes are differently expressed between in vitro M cells and Caco-2 cells, and to determine whether S. Typhimurium and speG affect the transcriptomes of both cell types. In vitro M cells and Caco-2 cells were infected with wild-type (WT) S. Typhimurium, its ΔspeG mutant, or none for 1 h for RNA microarrays; the transcriptomes among the 6 pools were pairwisely compared. Genetic loci encoding scaffold (e.g., HSCHR7_CTG4_4, HSCHR9_CTG9_35), long noncoding RNA, membrane-associated protein (PITPNB), neuron-related proteins (OR8D1, OR10G9, and NTNG2), and transporter proteins (MICU2 and SLC28A1) were significantly upregulated in uninfected M cells compared with uninfected Caco-2 cells; and their encoding proteins are promising M-cell markers. Significantly upregulated HSCHR7_CTG4_4 of uninfected in vitro M cells were speG-independently downregulated by S. Typhimurium infection that is a remarkable change representing an important but unreported characteristic of M cells. The immune responses of in vitro M cells and Caco-2 cells can differ and reply on speG or not, with speG-dependent regulation of KYL4, SCTR, IL6, TNF, and CELF4 in Caco-2 cells, JUN, KLF6, and KCTD11 in M cells, or speG-independent modulation of ZFP36 in both cells. This study facilitates understanding of the immune responses of in vitro M cells after administering the S. Typhimurium ΔspeG mutant as a future vaccine vector. PMID:27064787

  16. Oral insulin stimulates intestinal epithelial cell turnover following massive small bowel resection in a rat and a cell culture model.

    Science.gov (United States)

    Ben Lulu, Shani; Coran, Arnold G; Shehadeh, Naim; Shamir, Raanan; Mogilner, Jorge G; Sukhotnik, Igor

    2012-02-01

    We have recently reported that oral insulin (OI) stimulates intestinal adaptation after bowel resection and that OI enhances enterocyte turnover in correlation with insulin receptor expression along the villus-crypt axis. The purpose of the present study was to evaluate the effect of OI on intestinal epithelial cell proliferation and apoptosis in a rat model of short bowel syndrome (SBS) and in a cell culture model. Caco-2 cells were incubated with increasing concentrations of insulin. Cell proliferation and apoptosis were determined by FACS cytometry. Cell viability was investigated using the Alamar Blue technique. Male rats were divided into three groups: Sham rats underwent bowel transection, SBS rats underwent a 75% bowel resection, and SBS-OI rats underwent bowel resection and were treated with OI given in drinking water (1 U/ml) from the third postoperative day. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined on day 15. Real time PCR was used to determine the level of bax and bcl-2 mRNA and western blotting was used to determine bax, bcl-2, p-ERK and AKT protein levels. Statistical analysis was performed using the one-way ANOVA test, with P cells with insulin resulted in a significant increase in cell proliferation (twofold increase after 24 h and 37% increase after 48 h) and cell viability (in a dose-dependent manner), but did not change cell apoptosis. In a rat model of SBS, treatment with OI resulted in a significant increase in all parameters of intestinal adaptation. Elevated cell proliferation rate in insulin treated rats was accompanied by elevated AKT and p-ERK protein levels. Decreased cell apoptosis in SBS-INS rats corresponded with a decreased bax/bcl-2 ratio. Oral insulin stimulates intestinal epithelial cell turnover after massive small bowel resection in a rat model of SBS and a cell culture model.

  17. SIRT1 inhibits the mouse intestinal motility and epithelial proliferation

    Science.gov (United States)

    SIRT1 inhibits the mouse intestinal motility and epithelial proliferation. Sirtuin 1 (SIRT1), a NAD+-dependent histone deacetylase, is involved in a wide array of cellular processes, including glucose homeostasis, energy metabolism, proliferation and apoptosis, and immune response. However, it is un...

  18. Polyunsaturated fatty acid metabolism in enterocyte models: T84 cell line vs. Caco-2 cell line.

    Science.gov (United States)

    Beguin, Pauline; Schneider, Anne-Catherine; Mignolet, Eric; Schneider, Yves-Jacques; Larondelle, Yvan

    2014-02-01

    Human colon carcinoma cell lines such as Caco-2 cells, model of mature enterocytes and T84 cells, model of crypt cells are useful to study interactions between nutrient processing and metabolic functions at intestinal level. Our study aimed at comparing the ability of Caco-2 and T84 cells (1) to incorporate dietary polyunsaturated fatty acids (PUFA), (2) to process them and (3) to sort them into neutral lipids (NL), free fatty acids (FFA) and phospholipids (PL). Caco-2 and T84 cells were exposed to a 7-day long supplementation with PUFA. The amounts of fatty acids accumulated and incorporated into the NL, FFA or PL fractions were higher in Caco-2 than in T84 cells. Caco-2 cells were able to significantly elongate C18 PUFA and C20 PUFA of both n-3 and n-6 families. In contrast, T84 cells were unable to elongate the n-6 fatty acids whereas elongation of n-3 fatty acids was detectable but marginal. Similarly, a Δ6 desaturase activity was observed in Caco-2 but not in T84 cells. In T84 cells, each exogenous fatty acid was predominantly accumulated in the PL fraction. In Caco-2 cells, C20 fatty acids and C18:2n-6 was preferentially accumulated in the PL fraction, while C22 PUFA and C18:3n-3 was preferentially accumulated in the NL fraction. Overall, this study has shown that Caco-2 and T84 cells, as models of intestinal mucosal cells, present large differences in PUFA accumulation capacity, specific elongase and desaturase activities and distribution pattern of exogenous PUFA and of their metabolites in the lipid classes.

  19. Epithelial calcineurin controls microbiota-dependent intestinal tumor development

    Science.gov (United States)

    Peuker, Kenneth; Muff, Stefanie; Wang, Jun; Künzel, Sven; Bosse, Esther; Zeissig, Yvonne; Luzzi, Giuseppina; Basic, Marijana; Strigli, Anne; Ulbricht, Andrea; Kaser, Arthur; Arlt, Alexander; Chavakis, Triantafyllos; van den Brink, Gijs R; Schafmayer, Clemens; Egberts, Jan-Hendrik; Becker, Thomas; Bianchi, Marco E; Bleich, André; Röcken, Christoph; Hampe, Jochen; Schreiber, Stefan; Baines, John F; Blumberg, Richard S; Zeissig, Sebastian

    2017-01-01

    Inflammation-associated pathways are active in intestinal epithelial cells (IECs) and contribute to the pathogenesis of colorectal cancer (CRC). Calcineurin, a phosphatase required for the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors, shows increased expression in CRC. We therefore investigated the role of calcineurin in intestinal tumor development. We demonstrate that calcineurin and NFAT factors are constitutively expressed by primary IECs and selectively activated in intestinal tumors as a result of impaired stratification of the tumor-associated microbiota and toll-like receptor signaling. Epithelial calcineurin supports the survival and proliferation of cancer stem cells in an NFAT-dependent manner and promotes the development of intestinal tumors in mice. Moreover, somatic mutations that have been identified in human CRC are associated with constitutive activation of calcineurin, whereas nuclear translocation of NFAT is associated with increased death from CRC. These findings highlight an epithelial cell–intrinsic pathway that integrates signals derived from the commensal microbiota to promote intestinal tumor development. PMID:27043494

  20. Heat-Labile Enterotoxin-Induced PERK-CHOP Pathway Activation Causes Intestinal Epithelial Cell Apoptosis

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    Xi Lu

    2017-06-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC is a leading cause of diarrhea among children and travelers in developing countries, and heat-labile enterotoxin (LT is one of the most important virulence factors. The pathogenesis of and virulence factors associated with ETEC have been well-characterized; however, the extent to which ETEC damages host cells remains unclear. In this study, we found that LT could induce decreases in intestinal epithelial cell viability and induce apoptosis in a dose- and time- dependent manner in both HCT-8 and Caco-2 cells. We analyzed the expression profiles of apoptosis-related proteins via protein array technology and found that Bax, p-p53(S46, cleaved caspase-3, and TNFRI/TNFRSF1A expression levels were significantly up-regulated in wild-type ETEC- but not in ΔLT ETEC-infected HCT-8 cells. Bax is essential for endoplasmic reticulum (ER stress-triggered apoptosis, and our RNAi experiments showed that the PERK-eIF2-CHOP pathway and reactive oxygen species (ROS are also main participants in this process. LT-induced ROS generation was decreased in CHOP-knockdown HCT-8 cells compared to that in control cells. Moreover, pretreatment with the ROS inhibitor NAC down-regulated GRP78, CHOP, Bim, and cleaved caspase-3 expression, resulting in a reduction in the apoptosis rate from 36.2 to 20.3% in LT-treated HCT-8 cells. Furthermore, ROS inhibition also attenuated LT-induced apoptosis in the small intestinal mucosa in the ETEC-inoculation mouse model.

  1. Lactobacillus acidophilus alleviates platelet-activating factor-induced inflammatory responses in human intestinal epithelial cells.

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    Alip Borthakur

    Full Text Available Probiotics have been used as alternative prevention and therapy modalities in intestinal inflammatory disorders including inflammatory bowel diseases (IBD and necrotizing enterocolitis (NEC. Pathophysiology of IBD and NEC includes the production of diverse lipid mediators, including platelet-activating factor (PAF that mediate inflammatory responses in the disease. PAF is known to activate NF-κB, however, the mechanisms of PAF-induced inflammation are not fully defined. We have recently described a novel PAF-triggered pathway of NF-κB activation and IL-8 production in intestinal epithelial cells (IECs, requiring the pivotal role of the adaptor protein Bcl10 and its interactions with CARMA3 and MALT1. The current studies examined the potential role of the probiotic Lactobacillus acidophilus in reversing the PAF-induced, Bcl10-dependent NF-κB activation and IL-8 production in IECs. PAF treatment (5 µM×24 h of NCM460 and Caco-2 cells significantly increased nuclear p65 NF-κB levels and IL-8 secretion (2-3-fold, P<0.05, compared to control, which were blocked by pretreatment of the cells for 6 h with L. acidophilus (LA or its culture supernatant (CS, followed by continued treatments with PAF for 24 h. LA-CS also attenuated PAF-induced increase in Bcl10 mRNA and protein levels and Bcl10 promoter activity. LA-CS did not alter PAF-induced interaction of Bcl10 with CARMA3, but attenuated Bcl10 interaction with MALT1 and also PAF-induced ubiquitination of IKKγ. Efficacy of bacteria-free CS of LA in counteracting PAF-induced inflammatory cascade suggests that soluble factor(s in the CS of LA mediate these effects. These results define a novel mechanism by which probiotics counteract PAF-induced inflammation in IECs.

  2. Osmosignaling and volume regulation in intestinal epithelial cells.

    Science.gov (United States)

    Lim, Christina H; Bot, Alice G M; de Jonge, Hugo R; Tilly, Ben C

    2007-01-01

    Most cells have to perform their physiological functions under a variable osmotic stress, which, because of the relatively high permeability of the plasma membrane for water, may result in frequent alterations in cell size. Intestinal epithelial cells are especially prone to changes in cell volume because of their high capacity of salt and water transport and the high membrane expression of various nutrient transporters. Therefore, to avoid excessive shrinkage or swelling, enterocytes, like most cell types, have developed efficient mechanisms to maintain osmotic balance. This chapter reviews selected model systems that can be used to investigate cell volume regulation in intestinal epithelial cells, with emphasis on the regulatory volume decrease, and the methods available to study the compensatory redistribution of (organic) osmolytes. In addition, a brief summary is presented of the pathways involved in osmosensing and osmosignaling in the intestine.

  3. Fish oil enhances recovery of intestinal microbiota and epithelial integrity in chronic rejection of intestinal transplant.

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    Qiurong Li

    Full Text Available BACKGROUND: The intestinal chronic rejection (CR is the major limitation to long-term survival of transplanted organs. This study aimed to investigate the interaction between intestinal microbiota and epithelial integrity in chronic rejection of intestinal transplantation, and to find out whether fish oil enhances recovery of intestinal microbiota and epithelial integrity. METHODS/PRINCIPAL FINDINGS: The luminal and mucosal microbiota composition of CR rats were characterized by DGGE analysis at 190 days after intestinal transplant. The specific bacterial species were determined by sequence analysis. Furthermore, changes in the localization of intestinal TJ proteins were examined by immunofluorescent staining. PCR-DGGE analysis revealed that gut microbiota in CR rats had a shift towards Escherichia coli, Bacteroides spp and Clostridium spp and a decrease in the abundance of Lactobacillales bacteria in the intestines. Fish oil supplementation could enhance the recovery of gut microbiota, showing a significant decrease of gut bacterial proportions of E. coli and Bacteroides spp and an increase of Lactobacillales spp. In addition, CR rats showed pronounced alteration of tight junction, depicted by marked changes in epithelial cell ultrastructure and redistribution of occuldin and claudins as well as disruption in TJ barrier function. Fish oil administration ameliorated disruption of epithelial integrity in CR, which was associated with an improvement of the mucosal structure leading to improved tight junctions. CONCLUSIONS/SIGNIFICANCE: Our study have presented novel evidence that fish oil is involved in the maintenance of epithelial TJ integrity and recovery of gut microbiota, which may have therapeutic potential against CR in intestinal transplantation.

  4. Epithelial Cell Inflammasomes in Intestinal Immunity and Inflammation

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    Andrea C. Lei-Leston

    2017-09-01

    Full Text Available Pattern recognition receptors (PRR, such as NOD-like receptors (NLRs, sense conserved microbial signatures, and host danger signals leading to the coordination of appropriate immune responses. Upon activation, a subset of NLR initiate the assembly of a multimeric protein complex known as the inflammasome, which processes pro-inflammatory cytokines and mediates a specialized form of cell death known as pyroptosis. The identification of inflammasome-associated genes as inflammatory bowel disease susceptibility genes implicates a role for the inflammasome in intestinal inflammation. Despite the fact that the functional importance of inflammasomes within immune cells has been well established, the contribution of inflammasome expression in non-hematopoietic cells remains comparatively understudied. Given that intestinal epithelial cells (IEC act as a barrier between the host and the intestinal microbiota, inflammasome expression by these cells is likely important for intestinal immune homeostasis. Accumulating evidence suggests that the inflammasome plays a key role in shaping epithelial responses at the host–lumen interface with many inflammasome components highly expressed by IEC. Recent studies have exposed functional roles of IEC inflammasomes in mucosal immune defense, inflammation, and tumorigenesis. In this review, we present the main features of the predominant inflammasomes and their effector mechanisms contributing to intestinal homeostasis and inflammation. We also discuss existing controversies in the field and open questions related to their implications in disease. A comprehensive understanding of the molecular basis of intestinal inflammasome signaling could hold therapeutic potential for clinical translation.

  5. Experimental Evaluation of the Transport Mechanisms of PoIFN-α in Caco-2 Cells

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    Xin Liu

    2017-11-01

    Full Text Available For the development of an efficient intestinal delivery system for Porcine interferon-α (PoIFN-α, the understanding of transport mechanisms of which in the intestinal cell is essential. In this study, we investigated the absorption mechanisms of PoIFN-α in intestine cells. Caco-2 cells and fluorescein isothiocyanate-labeled (FITC-PoIFN-α were used to explore the whole transport process, including endocytosis, intracellular trafficking, exocytosis, and transcytosis. Via various techniques, the transport pathways of PoIFN-α in Caco-2 cells and the mechanisms were clarified. Firstly, the endocytosis of PoIFN-α by Caco-2 cells was time, concentration and temperature dependence. And the lipid raft/caveolae endocytosis was the most likely endocytic pathway for PoIFN-α. Secondly, both Golgi apparatus and lysosome were involved in the intracellular trafficking of PoIFN-α. Thirdly, the treatment of indomethacin resulted in a significant decrease of exocytosis of PoIFN-α, indicating the participation of cyclooxygenase. Finally, to evaluate the efficiency of PoIFN-α transport, the transepithelial electrical resistance (TEER value was measured to investigate the tight junctional integrity of the cell monolayers. The fluorescence microscope results revealed that the transport of PoIFN-α across the Caco-2 cell monolayers was restricted. In conclusion, this study depicts a probable picture of PoIFN-α transport in Caco-2 cells characterized by non-specificity, partial energy-dependency and low transcytosis.

  6. Experimental Evaluation of the Transport Mechanisms of PoIFN-α in Caco-2 Cells.

    Science.gov (United States)

    Liu, Xin; Zheng, Sidi; Qin, Yue; Ding, Wenya; Tu, Yabin; Chen, Xingru; Wu, Yunzhou; Yanhua, Li; Cai, Xuehui

    2017-01-01

    For the development of an efficient intestinal delivery system for Porcine interferon-α (PoIFN-α), the understanding of transport mechanisms of which in the intestinal cell is essential. In this study, we investigated the absorption mechanisms of PoIFN-α in intestine cells. Caco-2 cells and fluorescein isothiocyanate-labeled (FITC)-PoIFN-α were used to explore the whole transport process, including endocytosis, intracellular trafficking, exocytosis, and transcytosis. Via various techniques, the transport pathways of PoIFN-α in Caco-2 cells and the mechanisms were clarified. Firstly, the endocytosis of PoIFN-α by Caco-2 cells was time, concentration and temperature dependence. And the lipid raft/caveolae endocytosis was the most likely endocytic pathway for PoIFN-α. Secondly, both Golgi apparatus and lysosome were involved in the intracellular trafficking of PoIFN-α. Thirdly, the treatment of indomethacin resulted in a significant decrease of exocytosis of PoIFN-α, indicating the participation of cyclooxygenase. Finally, to evaluate the efficiency of PoIFN-α transport, the transepithelial electrical resistance (TEER) value was measured to investigate the tight junctional integrity of the cell monolayers. The fluorescence microscope results revealed that the transport of PoIFN-α across the Caco-2 cell monolayers was restricted. In conclusion, this study depicts a probable picture of PoIFN-α transport in Caco-2 cells characterized by non-specificity, partial energy-dependency and low transcytosis.

  7. Glycomacropeptide Reduces Intestinal Epithelial Cell Barrier Dysfunction and Adhesion of Entero-Hemorrhagic and Entero-Pathogenic Escherichia coli in Vitro

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    Shane Feeney

    2017-10-01

    Full Text Available In recent years, the potential of glycosylated food components to positively influence health has received considerable attention. Milk is a rich source of biologically active glycoconjugates which are associated with antimicrobial, immunomodulatory, anti-adhesion, anti-inflammatory and prebiotic properties. Glycomacropeptide (GMP is the C-terminal portion of kappa-casein that is released from whey during cheese-making by the action of chymosin. Many of the biological properties associated with GMP, such as anti-adhesion, have been linked with the carbohydrate portion of the protein. In this study, we investigated the ability of GMP to inhibit the adhesion of a variety of pathogenic Escherichia coli strains to HT-29 and Caco-2 intestinal cell lines, given the importance of E. coli in causing bacterial gastroenteritis. GMP significantly reduced pathogen adhesion, albeit with a high degree of species specificity toward enteropathogenic E. coli (EPEC strains O125:H32 and O111:H2 and enterohemorrhagic E. coli (EHEC strain 12900 O157:H7. The anti-adhesive effect resulted from the interaction of GMP with the E. coli cells and was also dependent on GMP concentration. Pre-incubation of intestinal Caco-2 cells with GMP reduced pathogen translocation as represented by a decrease in transepithelial electrical resistance (TEER. Thus, GMP is an effective in-vitro inhibitor of adhesion and epithelial injury caused by E. coli and may have potential as a biofunctional ingredient in foods to improve gastrointestinal health.

  8. Comparative proteomic analysis of cell lines and scrapings of the human intestinal epithelium

    Directory of Open Access Journals (Sweden)

    Renes Johan

    2007-04-01

    Full Text Available Abstract Background In vitro models are indispensable study objects in the fields of cell and molecular biology, with advantages such as accessibility, homogeneity of the cell population, reproducibility, and growth rate. The Caco-2 cell line, originating from a colon carcinoma, is a widely used in vitro model for small intestinal epithelium. Cancer cells have an altered metabolism, making it difficult to infer their representativity for the tissue from which they are derived. This study was designed to compare the protein expression pattern of Caco-2 cells with the patterns of intestinal epithelial cells from human small and large intestine. HT-29 intestinal cells, Hep G2 liver cells and TE 671 muscle cells were included too, the latter two as negative controls. Results Two-dimensional gel electrophoresis was performed on each tissue and cell line protein sample. Principal component and cluster analysis revealed that global expression of intestinal epithelial scrapings differed from that of intestinal epithelial cell lines. Since all cultured cell lines clustered together, this finding was ascribed to an adaptation of cells to culture conditions and their tumor origin, and responsible proteins were identified by mass spectrometry. When investigating the profiles of Caco-2 cells and small intestinal cells in detail, a considerable overlap was observed. Conclusion Numerous proteins showed a similar expression in Caco-2 cells, HT-29 cells, and both the intestinal scrapings, of which some appear to be characteristic to human intestinal epithelium in vivo. In addition, several biologically significant proteins are expressed at comparable levels in Caco-2 cells and small intestinal scrapings, indicating the usability of this in vitro model. Caco-2 cells, however, appear to over-express as well as under-express certain proteins, which needs to be considered by scientists using this cell line. Hence, care should be taken to prevent misinterpretation of

  9. Alternative Functional In Vitro Models of Human Intestinal Epithelia

    Directory of Open Access Journals (Sweden)

    Amanda L Kauffman

    2013-07-01

    Full Text Available Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We sought to evaluate and compare two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs and induced pluripotent stem cell (iPSC-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, our previously described 3-dimensional intestinal organogenesis method was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

  10. Assessment of cell viability and permeation enhancement in presence of lipid-based self-emulsifying drug delivery systems using Caco-2 cell model: Polysorbate 80 as the surfactant.

    Science.gov (United States)

    Bu, Pengli; Ji, Yue; Narayanan, Silpa; Dalrymple, Damon; Cheng, Xingguo; Serajuddin, Abu T M

    2017-03-01

    Lipid-based self-emulsifying drug delivery systems (SEDDS) are commonly used for solubilizing and enhancing oral bioavailability of poorly water-soluble drugs. However, their effects on viability of intestine epithelial cells and influence on membrane permeation are poorly understood. The present study was undertaken for safety assessment of lipid-based formulations containing medium-chain fatty acid esters as lipids and polysorbate 80 as the surfactant using the Caco-2 in vitro model. Any possible paracellular permeation enhancement through Caco-2 monolayers by the nontoxic formulations was also investigated. Mixtures of monoglyceride (Capmul MCM EP or 708G) or propylene glycol monoester (Capmul PG-8 NF) of medium chain fatty acids with polysorbate 80, with and without the incorporation of a medium-chain triglyceride (Captex 355), were prepared. After suitable dilution with aqueous culture medium, the formulations were incubated with a series of Caco-2 cultures of different maturity. Cell viability and membrane integrity were assessed. Any effects of nontoxic formulations on the transport of the fluorescent dye, Lucifer yellow, through Caco-2 monolayers were also determined. Formulations containing 1:1 ratios of monoglyceride or propylene glycol monoester to triglyceride (30% polysorbate 80, 35% monoglyceride or monoester and 35% triglyceride) were best tolerated by Caco-2 cells. Increased maturity obtained through longer culture durations rendered Caco-2 cells greater tolerance towards lipid-based formulations, and maximum tolerance to lipid-based formulations was observed with Caco-2 monolayers after being cultured for 21-23days. Furthermore, extent of cell membrane rupture caused by lipid-surfactant mixtures correlated positively with levels of cytotoxicity, suggesting a potential underlying mechanism. Permeation studies using Caco-2 monolayer model revealed that certain formulations significantly enhanced paracellular transport activities. Lipid-based SEDDS

  11. Competition of Lactobacillus paracasei with Salmonella enterica for Adhesion to Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Alicja Jankowska

    2008-01-01

    Full Text Available Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. In this study, we examined the ability of Lactobacillus paracasei to inhibit the adhesion of pathogenic Salmonella enterica to human colon adenocarcinoma Caco-2 cells. Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively. In adhesion experiments, bacteria were added to the cells for 2 or 4 hours. The number of attached bacteria was expressed as colony-forming units (CFUs, Caco-2 cells were counted in hematocytometer. Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to . paracasei and almost 27-fold higher after 4 hours of contact. Two types of experiments were done: coincubation (both bacteria were added to Caco-2 cells simultaneously, and preincubation (. paracasei was incubated with Caco-2 cells first, and then . enterica was added. In coincubation experiment, the presence of . paracasei decreased . enterica adhesion by 4-fold and in preincubation experiment even 7-fold. Generally, Lactobacillus spent culture supernatants (SCSs acted weaker as inhibitors of Salmonella adhesion in comparison to the whole . paracasei culture in coincubation experiment. In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation.

  12. Insights into Caco-2 cell culture structure using coherent anti-Stokes Raman scattering (CARS) microscopy.

    Science.gov (United States)

    Saarinen, Jukka; Sözeri, Erkan; Fraser-Miller, Sara J; Peltonen, Leena; Santos, Hélder A; Isomäki, Antti; Strachan, Clare J

    2017-05-15

    We have used coherent anti-Stokes Raman scattering (CARS) microscopy as a novel and rapid, label-free and non-destructive imaging method to gain structural insights into live intestinal epithelial cell cultures used for drug permeability testing. Specifically we have imaged live Caco-2 cells in (bio)pharmaceutically relevant conditions grown on membrane inserts. Imaging conditions were optimized, including evaluation of suitable membrane materials and media solutions, as well as tolerable laser powers for non-destructive imaging of the live cells. Lipid structures, in particular lipid droplets, were imaged within the cells on the insert membranes. The size of the individual lipid droplets increased substantially over the 21-day culturing period up to approximately 10% of the volume of the cross section of individual cells. Variation in lipid content has important implications for intestinal drug permeation testing during drug development but has received limited attention to date due to a lack of suitable analytical techniques. CARS microscopy was shown to be well suited for such analysis with the potential for in situ imaging of the same individual cell-cultures that are used for permeation studies. Overall, the method may be used to provide important information about cell monolayer structure to better understand drug permeation results. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Nitric oxide-induced hyperpermeability of human intestinal epithelial monolayers is augmented by inhibition of the amiloride-sensitive Na(+)-H+ antiport: potential role of peroxynitrous acid.

    Science.gov (United States)

    Unno, N; Menconi, M J; Fink, M P

    1997-08-01

    Nitric oxide (NO.) increases the permeability of cultured intestinal epithelial monolayers. NO. reacts with superoxide anion to form peroxynitrite anion, which can be protonated under mildly acidic conditions to form the potent and versatile oxidizing agent, peroxynitrous acid. We hypothesized that intracellular acidosis induced by the Na(+)-H+ antiport blocker, amiloride, would favor the formation of peroxynitrous acid and thereby augment hyperpermeability induced by the NO. donor, SIN-1. Caco-2BBe human intestinal epithelial monolayers were grown on permeable supports in bicameral chambers. The permeability of monolayers was assessed by measuring the transepithelial flux of fluorescein disulfonic acid (FS). Incubation of monolayers with SIN-1 increased permeability to FS. Adding amiloride augmented SIN-1-induced hyperpermeability. SIN-1 plus amiloride also decreased cellular adenosine triphosphate content and caused derangements of the actin-based cytoskeleton as demonstrated by fluorescence microscopy. Coincubation of monolayers with several free-radical or peroxynitrous acid scavengers (deferoxamine, mannitol, dimethyl sulfoxide, or ascorbate) ameliorated hyperpermeability induced by SIN-1 plus amiloride. Amiloride augments NO.-induced intestinal epithelial permeability, apparently by promoting the development of intracellular acidosis and thereby favoring the formation of the peroxynitrous acid.

  14. Effect of Utipro(®) (containing gelatin-xyloglucan) against Escherichia coli invasion of intestinal epithelial cells: results of an in vitro study.

    Science.gov (United States)

    de Servi, Barbara; Ranzini, Francesco; Piqué, Nuria

    2016-05-01

    To evaluate whether Utipro(®), a natural product approved to prevent urinary tract infections, protects intestinal epithelial cells from Escherichia coli adherence/intracellular invasion in vitro. Caco-2 and CacoGoblet(TM) cells were treated with Utipro (1.5 to 10 mg/ml) or untreated (controls). E. coli adherence/intracellular invasion was evaluated by Trans-Epithelial Electrical Resistance, Lucifer Yellow assay and microbial counts. Utipro was noncytotoxic. Utipro 5 and 10 mg/ml protected cell tight junctions (mean ± SD Trans-Epithelial Electrical Resistance [Ω × cm(2)] 66.83 ± 0.29 and 71.33 ± 0.29, respectively), and protected cells from E. coli intracellular invasion (mean ± SD reductions in total bacteria counts [Log10] 0.9 ± 0.06 and 2.1 ± 0.56, respectively). Results indicate that Utipro creates a protective physical barrier on intestinal epithelial cells in vitro which reduces the settling of E. coli reservoirs. These results constitute the first step in the demonstration of the efficacy of Utipro to prevent urinary tract infections. Further research is needed in in vivo models and clinical trials.

  15. Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides.

    Science.gov (United States)

    Bocsik, Alexandra; Walter, Fruzsina R; Gyebrovszki, Andrea; Fülöp, Lívia; Blasig, Ingolf; Dabrowski, Sebastian; Ötvös, Ferenc; Tóth, András; Rákhely, Gábor; Veszelka, Szilvia; Vastag, Monika; Szabó-Révész, Piroska; Deli, Mária A

    2016-02-01

    The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  16. Mitochondrial ATP depletion disrupts Caco-2 monolayer integrity and internalizes claudin 7

    NARCIS (Netherlands)

    Janssen Duijghuijsen, Lonneke M.; Grefte, Sander; Boer, de Vincent C.J.; Zeper, Lara; Dartel, van Dorien A.M.; Romijnders-van der Stelt, Inge; Bekkenkamp-Grovenstein, Melissa; Norren, van Klaske; Wichers, Harry J.; Keijer, Jaap

    2017-01-01

    Objective: In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Methods: Human Caco-2 cells were cultured for 10 days in culture

  17. Interactions between organic anions on multiple transporters in Caco-2 cells

    DEFF Research Database (Denmark)

    Grandvuinet, Anne Sophie; Steffansen, Bente

    2011-01-01

    Caco-2 cell line may be used as an overall model to predict interactions on multiple membrane transporters in the intestine. Taurocholic acid (TCA) and estrone-3-sulfate (E1S) were used as model substrates. Possible inhibitors studied were TCA, E1S, taurolithocholic acid, fluvastatin, and glipizide...

  18. Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Banerjee Pratik

    2009-04-01

    Full Text Available Abstract Background Probiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD. Clostridium difficile is the most common cause of AAD and the resulting C. difficile – mediated infection (CDI, is potentially deadly. C. difficile associated diarrhea (CDAD is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by C. difficile causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892 on C. difficile-mediated cytotoxicity using Caco-2 cells as a model. Methods Experiments were carried out to test if the cytotoxicity induced by C. difficile-conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS from LDB B-30892 in different dilutions (1:2 to 1:2048. In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect C. difficile induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of C. difficile to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on C. difficile. Results and discussion Co-culturing of LDB B-30892 with C. difficile inhibited the C. difficile-mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892-C. difficile co-culture was administered (up to a dilution of 1:16 on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin

  19. Effect of Maillard reaction on biochemical properties of peanut 7S globulin (Ara h 1) and its interaction with a human colon cancer cell line (Caco-2).

    Science.gov (United States)

    Teodorowicz, Małgorzata; Fiedorowicz, Ewa; Kostyra, Henryk; Wichers, Harry; Kostyra, Elżbieta

    2013-12-01

    The purpose of this study was to determine the influence of Maillard reaction (MR, glycation) on biochemical and biological properties of the major peanut allergen Ara h 1. Three different time/temperature conditions of treatment were applied (37, 60, and 145 °C). The extent of MR was assessed by SDS-PAGE, loss of free amino groups, fluorescence intensity, content of bound sugar and fructosamine. The Caco-2 model system was applied to study effects of hydrolysed and non-hydrolysed Ara h 1 on proliferation and interleukin-8 (IL-8) secretion from Caco-2 cells. We demonstrated significant differences in the biochemical properties of Ara h 1 glycated at different time/temperature conditions. Glycation of Ara h 1 at 37 °C was shown to cause least biochemical changes, not limiting pepsin hydrolysis. Loss of free amino groups, increase of fluorescence and brown colour of Ara h 1 glycated at 60 and 145 °C indicated advanced and final stages of MR. Non-treated Ara h 1 inhibited Caco-2 cell proliferation and stimulated IL-8 secretion. This effect was less pronounced for glycated Ara h 1. Incubation of Caco-2 cells with non-hydrolysed Ara h 1, glycated at the temperature of 37 and 60 °C, did not stimulate IL-8 secretion. Each applied time/temperature-treatment combination caused different biochemical changes of Ara h 1, underlining diversity of formed MRPs. MR, independently of temperature/time conditions, reduced the pro-inflammatory properties of native Ara h 1, reflected in stimulation of IL-8 secretion from intestinal epithelial cells.

  20. A Model of Cadmium Uptake and Transport in Caco-2 Cells.

    Science.gov (United States)

    Gerasimenko, T N; Senyavina, N V; Anisimov, N U; Tonevitskaya, S A

    2016-05-01

    We created a physiologically substantiated kinetic model of cadmium transport and toxicity in intestinal cell model (Caco-2 cells). Transcriptome profiling of Caco-2 cells revealed high content of transporter DMT1 and ZIP14 and intensive expression of some calcium channels of the CACN family. The mathematical model describing three types of transporters, as well as intracellular cadmium binding with metallothionein and excretion through the basolateral membrane allowed us to construct cadmium uptake and transport curves that approximated the previously obtained experimental data. Using the proposed model, we determined toxic intracellular cadmium concentration leading to cell death and impairing the integrity of cell monolayer and described cadmium transport in this case.

  1. Complete genome sequence of Bifidobacterium animalis subsp. lactis KLDS 2.0603, a probiotic strain with digestive tract resistance and adhesion to the intestinal epithelial cells.

    Science.gov (United States)

    Zhu, Dequan; Sun, Yu; Huo, Gui-Cheng; Yang, Limei; Liu, Fei; Li, Aili; Meng, Xiang-Chen

    2016-02-20

    Bifidobacterium animalis subsp. lactis KLDS 2.0603 (abbreviated as KLDS 2.0603) is a probiotic strain isolated from the feces of an adult human. Previous studies showed that KLDS 2.0603 has a high resistance to simulated digestive tract conditions and a high ability to adhere to intestinal epithelial cells (Caco-2). These two characteristics are essential requirements for the selection of probiotic bacteria. To explore the stress resistance mechanism to the digestive tract environment and the adhesive proteins of this strain, in this paper, we reported the complete genome sequence of KLDS 2.0603, which contains 19,469bp and encodes 1614 coding sequences(CDSs), 15 rRNA genes, 52 tRNA genes with 1678 open reading frames. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Complete genome sequence of bacteriocin-producing Lactobacillus plantarum KLDS1.0391, a probiotic strain with gastrointestinal tract resistance and adhesion to the intestinal epithelial cells.

    Science.gov (United States)

    Jia, Fang-Fang; Zhang, Lu-Ji; Pang, Xue-Hui; Gu, Xin-Xi; Abdelazez, Amro; Liang, Yu; Sun, Si-Rui; Meng, Xiang-Chen

    2017-10-01

    Lactobacillus plantarum KLDS1.0391 is a probiotic strain isolated from the traditional fermented dairy products and identified to produce bacteriocin against Gram-positive and Gram-negative bacteria. Previous studies showed that the strain has a high resistance to gastrointestinal stress and has a high adhesion ability to the intestinal epithelial cells (Caco-2). We reported the entire genome sequence of this strain, which contains a circular 2,886,607-bp chromosome and three circular plasmids. Genes, which are related to the biosynthesis of bacteriocins, the stress resistance to gastrointestinal tract environment and adhesive performance, were identified. Whole genome sequence of Lactobacillus plantarum KLDS1.0391 will be helpful for its applications in food industry. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Biosynthesis and transport of lysosomal alpha-glucosidase in the human colon carcinoma cell-line Caco-2: secretion from the apical surface

    NARCIS (Netherlands)

    Klumperman, J.; Fransen, J.A.; Boekestijn, J.C.; Oude Elferink, R.P.; Matter, K.; Hauri, H.P.; Tager, J.M.; Ginsel, L.A.

    1991-01-01

    The human adenocarcinoma cell line Caco-2 was used for studies on the biosynthesis and transport of lysosomal acid alpha-glucosidase in polarized epithelial cells. Metabolic labelling revealed that in Caco-2 cells alpha-glucosidase is synthesized as a precursor form of 110 x 10(3) Mr. This form is

  4. Biosynthesis and transport of lysosomal alpha-glucosidase in the human colon carcinoma cell line Caco-2: secretion from the apical surface

    NARCIS (Netherlands)

    Klumperman, J.; Fransen, J. A.; Boekestijn, T. C.; Oude Elferink, R. P.; Matter, K.; Hauri, H. P.; Tager, J. M.; Ginsel, L. A.

    1991-01-01

    The human adenocarcinoma cell line Caco-2 was used for studies on the biosynthesis and transport of lysosomal acid alpha-glucosidase in polarized epithelial cells. Metabolic labelling revealed that in Caco-2 cells alpha-glucosidase is synthesized as a precursor form of 110 x 10(3) Mr. This form is

  5. SGLT1-Mediated Transport in Caco-2 Cells Is Highly Dependent on Cell Bank Origin.

    Science.gov (United States)

    Steffansen, Bente; Pedersen, Maria D L; Laghmoch, Abdel M; Nielsen, Carsten U

    2017-09-01

    The human colon adenocarcinoma (Caco-2) cell line is a well-established in vitro model for studying transport phenomena for prediction of intestinal nutrient and drug absorption. However, substances depending on transporters such predictions are complicated due to variable transporter expression and limited knowledge about transporter function during multiple cell passaging and cell thawings. In the case of sodium glucose transporter 1 (SGLT1), a key transporter of oral absorption of d-glucose, one reason for compromised prediction could be inadequate expression of SGLT1 in Caco-2 cells and thereby limited sensitivity in the determination of SGLT1-mediated permeability (PSGLT1). Here, the objective is to characterize and compare SGLT1-mediated uptake in Caco-2 cells obtained from different cell banks. SGLT1-mediated uptake of the standard SGLT1 substrate, methyl-α-d-glucopyranoside, in Caco-2 cells was shown to be highly dependent on cell bank origin. The most robust and reliable SGLT1 functionality was identified in Caco-2 cells from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ), whereas cells from the American Type Culture Collection and European Collection of Authenticated Cell Cultures have lower SGLT1 transport activity. Transepithelial PSGLT1 across Caco-2 cells from DSMZ showed that PSGLT1 likely accounts for approximately 97% of absorptive methyl-α-d-glucopyranoside Papp(a-b). In conclusion, Caco-2 cells from DSMZ provide a robust in vitro model for studying SGLT1-mediated uptake and transport-over multiple cell passages and independent cell stock thawings. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  6. Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine

    NARCIS (Netherlands)

    Klunder, Leon J.; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C. D.

    Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we

  7. Selected Phytochemicals and Culinary Plant Extracts Inhibit Fructose Uptake in Caco-2 Cells

    OpenAIRE

    Yurim Lee; Yeni Lim; Oran Kwon

    2015-01-01

    This study compared the ability of nine culinary plant extracts containing a wide array of phytochemicals to inhibit fructose uptake and then explored the involvement of intestinal fructose transporters and phytochemicals for selected samples. The chemical signature was characterized by high performance liquid chromatography with mass spectrometry. Inhibition of [14C]-fructose uptake was tested by using human intestinal Caco-2 cells. Then, the relative contribution of the two apical-facing i...

  8. Detection of Maillard Reaction Product [5-(5,6-Dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol (F3-A) in Breads and Demonstration of Bioavailability in Caco-2 Intestinal Cells.

    Science.gov (United States)

    Chen, Xiu-Min; Dai, Yue; Kitts, David D

    2016-11-30

    [5-(5,6-Dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol, also called F3-A, has been isolated from hexose-lysine Maillard reaction (MR) models. Here we report on optimized conditions for the recovery of F3-A and concentrations found in bread. Recovery of F3-A was best achieved when samples were extracted with dichloromethane (DCM) at a solvent to sample ratio of 2:1 (v/v) after adjustment of the pH to 12. The amount of F3-A in whole wheat bread was significantly (P F3-A (0.9-7.8 μg/100 g) than the bread crumb (not detectable-3.5 μg/100 g); and toasting increased F3-A concentration with a range of not detectable to 6.0 μg/100 g in the control bread and 4.0 and 17.7 μg/100 g in the dark-toasted white sandwich bread and 100% whole wheat sandwich bread, respectively. The in vitro permeability of F3-A was measured using Caco-2 cell monolayer. The apparent permeability coefficient (Papp) of F3-A is (6.01 ± 0.35) × 10(-5) cm/s, which is similar to that of propranolol, a highly passive transcellular absorbed drug. In conclusion, the concentration of F3-A recovered in bread varies with the type of bread and degree of toasting, and F3-A is bioavailable.

  9. Design of 3D printed insert for hanging culture of Caco-2 cells.

    Science.gov (United States)

    Shen, Chong; Meng, Qin; Zhang, Guoliang

    2014-12-17

    A Caco-2 cell culture on Transwell, an alternative testing to animal or human testing used in evaluating drug intestinal permeability, incorrectly estimated the absorption of actively transported drugs due to the low expression of membrane transporters. Similarly, three-dimensional (3D) cultures of Caco-2 cells, which have been recommended to be more physiological relevant, were not superior to the Transwell culture in either accuracy or convenience in drug permeability testing. Using rapid 3D printing prototyping techniques, this study proposed a hanging culture of Caco-2 cells that performed with high accuracy in predicting drug permeability in humans. As found, hanging cultured Caco-2 cells formed a confluent monolayer and maintained high cell viability on the 3D printed insert. Compared with the normal culture on Transwell, the Caco-2 cells on the 3D printed insert presented ∼30-100% higher brush border enzyme activity and ∼2-7 folds higher activity of P-glycoprotein/multidrug resistance-associated protein 2 during 21 days of incubation. For the eight membrane transporter substrates, the predictive curve of the 3D printing culture exhibited better linearity (R(2) = 0.92) to the human oral adsorption than that of the Transwell culture (R(2) = 0.84), indicating better prediction by the 3D printing culture. In this regard, the 3D printed insert for hanging culture could be potentially developed as a convenient and low-cost tool for testing drug oral absorption.

  10. HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells

    DEFF Research Database (Denmark)

    Jørgensen, Steffen; Coshun, Mehmet; Mikkelsen Homburg, Keld

    2016-01-01

    The mammalian Caudal-related homeobox transcription factor 2 (CDX2) plays a key role in the homeobox regulatory network and is essential in regulating the expression of several homeobox (HOX) genes during embryonic development, particularly in the gut. Genome-wide CDX2 chromatin immunoprecipitation...... analysis and expression data from Caco2 cells also suggests a role for CDX2 in the regulation of HOXB4 gene expression in the intestinal epithelium. Thus, the aim of this study was to investigate whether HOXB4 gene expression is regulated by CDX2 in the intestinal epithelium. We demonstrated binding of CDX...... demonstrating the CDX2 regulation of HOXB4 gene expression in the developed intestinal epithelium, indicating a possible role for HOXB4 in intestinal homeostasis....

  11. Transport of hop aroma compounds across Caco-2 monolayers.

    Science.gov (United States)

    Heinlein, A; Metzger, M; Walles, H; Buettner, A

    2014-11-01

    Although being reported and used as a sedative remedy for several years, the bioactive principle of hop preparations is still not decisively clarified. Understanding absorption and transformation processes of potential physiologically active constituents is essential to evaluate the likeliness of biological effects on humans. Therefore, single hop aroma compounds as well as digestive transformation products thereof have been investigated in view of their human intestinal absorption, applying Caco-2 transport experiments as well as investigations on potential biotransformation processes. Selective and sensitive identification and quantification were thereby achieved by application of two-dimensional high resolution gas chromatography-mass spectrometry in conjunction with stable isotope dilution analysis, leading to the determination of apparent permeability values by different mathematical approaches considering sink and non-sink conditions. Overall, calculated permeability values ranged from 2.6 × 10(-6) to 1.8 × 10(-4) cm s(-1) with all mathematical approaches, indicating high absorption potential and almost complete bioavailability for all tested compounds with hydroxyl-functionalities. Considering this high permeability together with the high lipophilicity of these substances, a passive transcellular uptake route can be speculated. Investigated sesquiterpenes and β-myrcene showed flat absorption profiles while the investigated esters showed decreasing profiles. In view of the lipophilic and volatile nature of the investigated substances, special attention was paid to recovery and mass balance determination. Furthermore, in the course of the transport experiments of 1-octen-3-ol and 3-methyl-2-buten-1-ol, additional biotransformation products were observed, namely 3-octanone and 3-methyl-2-butenal, respectively. The absence of these additional substances in control experiments strongly indicates an intestinal first-pass metabolism of the

  12. New insights into mycotoxin mixtures: The toxicity of low doses of Type B trichothecenes on intestinal epithelial cells is synergistic

    Energy Technology Data Exchange (ETDEWEB)

    Alassane-Kpembi, Imourana [INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, F-31027 Toulouse (France); Université de Toulouse, ENVT, INP, UMR 1331 Toxalim, F-31076 Toulouse (France); Institut des Sciences Biomédicales Appliquées, Cotonou, Bénin (Benin); Kolf-Clauw, Martine; Gauthier, Thierry; Abrami, Roberta [INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, F-31027 Toulouse (France); Université de Toulouse, ENVT, INP, UMR 1331 Toxalim, F-31076 Toulouse (France); Abiola, François A. [Institut des Sciences Biomédicales Appliquées, Cotonou, Bénin (Benin); Oswald, Isabelle P., E-mail: Isabelle.Oswald@toulouse.inra.fr [INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, F-31027 Toulouse (France); Université de Toulouse, ENVT, INP, UMR 1331 Toxalim, F-31076 Toulouse (France); Puel, Olivier [INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, F-31027 Toulouse (France); Université de Toulouse, ENVT, INP, UMR 1331 Toxalim, F-31076 Toulouse (France)

    2013-10-01

    Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. DON is often present with other type B trichothecenes such as 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). Although the cytotoxicity of individual mycotoxins has been widely studied, data on the toxicity of mycotoxin mixtures are limited. The aim of this study was to assess interactions caused by co-exposure to Type B trichothecenes on intestinal epithelial cells. Proliferating Caco-2 cells were exposed to increasing doses of Type B trichothecenes, alone or in binary or ternary mixtures. The MTT test and neutral red uptake, respectively linked to mitochondrial and lysosomal functions, were used to measure intestinal epithelial cytotoxicity. The five tested mycotoxins had a dose-dependent effect on proliferating enterocytes and could be classified in increasing order of toxicity: 3-ADON < 15-ADON ≈ DON < NIV ≪ FX. Binary or ternary mixtures also showed a dose-dependent effect. At low concentrations (cytotoxic effect between 10 and 30–40%), mycotoxin combinations were synergistic; however DON–NIV–FX mixture showed antagonism. At higher concentrations (cytotoxic effect around 50%), the combinations had an additive or nearly additive effect. These results indicate that the simultaneous presence of low doses of mycotoxins in food commodities and diet may be more toxic than predicted from the mycotoxins alone. Considering the frequent co-occurrence of trichothecenes in the diet and the concentrations of toxins to which consumers are exposed, this synergy should be taken into account. - Highlights: • We assessed the individual and combined cytotoxicity of five trichothecenes. • The tested concentrations correspond to the French consumer exposure levels. • The type of interaction in combined cytotoxicity varied with the effect level. • Low doses of Type B trichothecenes induced synergistic

  13. Development, validation, and application of a novel 7-day Caco-2 cell culture system.

    Science.gov (United States)

    Cai, Yike; Xu, Chenshu; Chen, Peiyi; Hu, Jinqing; Hu, Rong; Huang, Min; Bi, Huichang

    2014-01-01

    Monolayers of Caco-2 cells have been widely accepted as one of the well-established in vitro models to predict intestinal drug permeability and absorption in humans. However, the procedure for culturing the traditional 21-day Caco-2 model is labor intensive and time consuming, which limits its wide application in drug development. The objective of the present study was to develop a rapid Caco-2 model with a 7-day cell culture process. A few modifications of the BIOCOAT® HTS Caco-2 Assay System were introduced including changing the cell seeding density, the composition of cell culture media, and the interval to change media. The monolayer structure was visualized through confocal microscopy. The transepithelial electrical resistance (TEER), apparent permeability coefficients (Papp), and functional activity of P-glycoprotein (P-gp) were determined and compared with the 21-day model. Moreover, pharmacokinetic studies were performed in animal models to evaluate the absolute bioavailability of oral doses (Foral) of different compounds. Our newly developed 7-day Caco-2 model displayed comparable cellular morphology and integrity with the traditional 21-day model. No significant difference in paracellular and transcellular permeability was observed between the two systems. The efflux ratios of transporting digoxin, the prototypical substrate of P-gp in 21- and 7-day systems were 16.04 and 24.92, respectively. When the P-gp inhibitor verapamil was present, the efflux ratios of 21- and 7-day systems were 1.37 and 0.86, respectively, suggesting the comparability of the P-gp functional activity in both systems. Furthermore, pharmacokinetic studies of several compounds performed in animal models revealed that the absolute bioavailability of oral doses in vivo was well correlated with the Caco-2 permeability in vitro. The novel system provides a rapid and economical option for assessing the drug permeability, and is applicable to the studies of intestinal drug absorption

  14. The Inside Story of Shigella Invasion of Intestinal Epithelial Cells

    Science.gov (United States)

    Carayol, Nathalie; Tran Van Nhieu, Guy

    2013-01-01

    As opposed to other invasive pathogens that reside into host cells in a parasitic mode, Shigella, the causative agent of bacillary dysentery, invades the colonic mucosa but does not penetrate further to survive into deeper tissues. Instead, Shigella invades, replicates, and disseminates within the colonic mucosa. Bacterial invasion and spreading in intestinal epithelium lead to the elicitation of inflammatory responses responsible for the tissue destruction and shedding in the environment for further infection of other hosts. In this article, we highlight specific features of the Shigella arsenal of virulence determinants injected by a type III secretion apparatus (T3SA) that point to the targeting of intestinal epithelial cells as a discrete route of invasion during the initial event of the infectious process. PMID:24086068

  15. Epithelial sphingolipid sorting allows for extensive variation of the fatty acyl chain and the sphingosine backbone

    NARCIS (Netherlands)

    van 't Hof, W.J.; Silvius, J.R.; Wieland, F.T.; van Meer, G.|info:eu-repo/dai/nl/068570368

    1992-01-01

    In kidney MDCK and intestinal Caco-2 epithelial cells, glucosylceramide (GlcCer) and sphingomyelin (SPH) synthesized from the short-chain sphingolipid analogue N-6-[7-nitro-2,1,3-benzoxadiazol-4-yl]aminodecanoyl (C6-NBD)-ceramide are delivered to the cell surface with apical/basolateral polarities

  16. Enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model.

    Science.gov (United States)

    Naz, Neveda; Mills, Dominic C; Wren, Brendan W; Dorrell, Nick

    2013-10-22

    The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

  17. Permeability of rosmarinic acid in Prunella vulgaris and ursolic acid in Salvia officinalis extracts across Caco-2 cell monolayers.

    Science.gov (United States)

    Qiang, Zhiyi; Ye, Zhong; Hauck, Cathy; Murphy, Patricia A; McCoy, Joe-Ann; Widrlechner, Mark P; Reddy, Manju B; Hendrich, Suzanne

    2011-10-11

    Rosmarinic acid (RA), a caffeic acid-related compound found in high concentrations in Prunella vulgaris (self-heal), and ursolic acid (UA), a pentacyclic triterpene acid concentrated in Salvia officinalis (sage), have been traditionally used to treat inflammation in the mouth, and may also be beneficial for gastrointestinal health in general. To investigate the permeabilities of RA and UA as pure compounds and in Prunella vulgaris and Salvia officinalis ethanol extracts across human intestinal epithelial Caco-2 cell monolayers. The permeabilities and phase II biotransformation of RA and UA as pure compounds and in herbal extracts were compared using Caco-2 cells with HPLC detection. The apparent permeability coefficient (P(app)) for RA and RA in Prunella vulgaris extracts was 0.2 ± 0.05 × 10(-6)cm/s, significantly increased to 0.9 ± 0.2 × 10(-6)cm/s after β-glucuronidase/sulfatase treatment. P(app) for UA and UA in Salvia officinalis extract was 2.7 ± 0.3 × 10(-6)cm/s and 2.3 ± 0.5 × 10(-6)cm/s before and after β-glucuronidase/sulfatase treatment, respectively. Neither compound was affected in permeability by the herbal extract matrix. RA and UA in herbal extracts had similar uptake as that found using the pure compounds, which may simplify the prediction of compound efficacy, but the apparent lack of intestinal glucuronidation/sulfation of UA is likely to further enhance the bioavailability of that compound compared with RA. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Gelatin tannate reduces the proinflammatory effects of lipopolysaccharide in human intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Frasca G

    2012-05-01

    Full Text Available Giuseppina Frasca1, Venera Cardile1, Carmelo Puglia2, Claudia Bonina2, Francesco Bonina21Department of Biomedical Sciences, (Physiology, 2Department of Drug Sciences, University of Catania, Catania, ItalyBackground: Gelatin tannate is a mixture of tannic acid and gelatin. Tannic acid has astringent properties, due to its capacity to form protein–macromolecular complexes, as well as antibacterial and antioxidant properties. However, little is known about its anti-inflammatory properties. Purpose: To evaluate the anti-inflammatory activity of gelatin tannate by quantifying the suppression of key molecules produced during inflammatory events in lipopolysaccharide (LPS-stimulated human intestinal cells. Methods: Intercellular adhesion molecule-1 (ICAM-1 expression was determined by Western blot analysis; interleukin-8 (IL-8 and tumor necrosis factor-α (TNF-α concentrations were measured by enzyme-linked immunosorbent assays in Caco-2 cells 24 hours after treatment with LPS (1 μg/mL in presence of different concentrations of gelatin tannate. Results: ICAM-1 is induced on a wide variety of cells by inflammatory stimuli such as LPS. Our results have shown gelatin tannate as a potent inhibitor of ICAM-1 expression in LPS-stimulated Caco-2 cells. IL-8 and TNF-α are important inflammatory mediators, recruiting neutrophils and T-lymphocytes. Together with LPS, adding gelatin tannate at different concentrations induced a dose-dependent inhibition of IL-8 and TNF-α released by Caco-2 cells. Conclusion: These results suggest that gelatin tannate exerts anti-inflammatory effects by inhibiting the specific cytokines and adhesion molecules involved in several inflammatory disorders.Keywords: Caco-2, ICAM-1, IL-8, TNF-α

  19. Molecular cloning, sequence analysis, and function of the intestinal epithelial stem cell marker Bmi1 in pig intestinal epithelial cells.

    Science.gov (United States)

    Li, C-M; Yan, H-C; Fu, H-L; Xu, G-F; Wang, X-Q

    2014-01-01

    In the present work, we cloned the full-length cDNA of the pig Bmi1 gene (BMI1 polycomb ring finger oncogene), which has been indicated as an intestinal epithelial stem cell (IESC) marker in other mammals. This paper provides the first report of the function of Bmi1 in pig intestinal epithelial cells and a brief description of its underlying mechanism. Rapid amplification of cDNA ends technology was used to clone the complete pig Bmi1 sequence, and a Bmi1-pcDNA3.1 vector was constructed for transfection into an intestinal porcine epithelial cell line (IPEC-1). The proliferation ability of the cells was estimated using the MTT assay and the EdU incorporation method at different time points after seeding. Cell cycle information was detected by flow cytometry. The mRNA abundances of cell cycle-related genes were also measured. The results indicated that the pig Bmi1 cDNA is 3,193 bp in length and consists of a 981 bp open reading frame, a 256 bp 5´ untranslated region (UTR), and a 1,956 bp 3' UTR. The transcript contains no signal peptides, and there are no transmembrane regions in the pig Bmi1 coded protein, which has a total of 326 AA. The overexpression of the pig Bmi1 in the IPEC-1 cells led to increased cell proliferation and a lower percentage of cells in the G1 and S phases (P cells in the G2 phase (P 0.05). Our data suggested that pig Bmi1 can increase the proliferation of IPEC-1 cells by promoting the G1/S transition and the overall cell cycle process.

  20. Monolayers of IEC-18 cells as an in vitro model for screening the passive transcellular and paracellular transport across the intestinal barrier: Comparison of active and passive transport with the human colon carcinoma Caco-2 cell line

    NARCIS (Netherlands)

    Versantvoort, C.H.M.; Ondrewater, R.C.A.; Duizer, E.; Sandt, J.J.M. van de; Gilde, A.J.; Groten, J.P.

    2002-01-01

    Purpose: previous studies have shown that the rat small intestinal cell line IEC-18 provides a size-selective barrier for paracellularly transported hydrophilic macromolecules. In order to determine the utility of IEC-18 cells as an in vitro model to screen the passive paracellular and transcellular

  1. Applications of a 7-day Caco-2 cell model in drug discovery and development.

    Science.gov (United States)

    Peng, Ying; Yadava, Preeti; Heikkinen, Aki T; Parrott, Neil; Railkar, Aruna

    2014-06-02

    Oral delivery is the preferred route of administration and therefore good absorption after oral dosing is a prerequisite for a compound to be successful in the clinic. The prediction of oral bioavailability from in vitro permeability assays is thus a valuable tool during drug discovery and development. Caco-2 cell monolayers mimic the human intestinal epithelium in many aspects. These monolayers form tight junctions between cells and have been widely used as a model of human intestinal absorption. Caco-2 cells also express a variety of transporter proteins although the transformed nature of the cells results in unpredictable differentiation markers, transport properties and enzyme expression. Thus various modifications of the Caco-2 assay are used in laboratories across the globe. The purpose of this paper is to provide an overview of a time and resource saving 7-day Caco-2 assay protocol. We also discuss the impact of various experimental conditions on permeability measurements and its applications during lead optimization in early discovery and for clinical candidate characterization, specifically for prediction of absorption in human, at a later stage in drug development. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Penetration of CdSe/ZnS quantum dots into differentiated vs undifferentiated Caco-2 cells.

    Science.gov (United States)

    Peuschel, Henrike; Ruckelshausen, Thomas; Kiefer, Silke; Silina, Yuliya; Kraegeloh, Annette

    2016-09-26

    Quantum dots (QDs) have great potential as fluorescent labels but cytotoxicity relating to extra- and intracellular degradation in biological systems has to be addressed prior to biomedical applications. In this study, human intestinal cells (Caco-2) grown on transwell membranes were used to study penetration depth, intracellular localization, translocation and cytotoxicity of CdSe/ZnS QDs with amino and carboxyl surface modifications. The focus of this study was to compare the penetration depth of QDs in differentiated vs undifferentiated cells using confocal microscopy and image processing. Caco-2 cells were exposed to QDs with amino (NH2) and carboxyl (COOH) surface groups for 3 days using a concentration of 45 µg cadmium ml-1. Image analysis of confocal/multiphoton microscopy z-stacks revealed no penetration of QDs into the cell lumen of differentiated Caco-2 cells. Interestingly, translocation of cadmium ions onto the basolateral side of differentiated monolayers was observed using high resolution inductively coupled plasma mass spectrometry (ICP-MS). Membrane damage was neither detected after short nor long term incubation in Caco-2 cells. On the other hand, intracellular localization of QDs after exposure to undifferentiated cells was observed and QDs were partially located within lysosomes. In differentiated Caco-2 monolayers, representing a model for small intestinal enterocytes, no penetration of amino and carboxyl functionalized CdSe/ZnS QDs into the cell lumen was detected using microscopy analysis and image processing. In contrast, translocation of cadmium ions onto the basolateral side could be detected using ICP-MS. However, even after long term incubation, the integrity of the cell monolayer was not impaired and no cytotoxic effects could be detected. In undifferentiated Caco-2 cells, both QD modifications could be found in the cell lumen. Only to some extend, QDs were localized in endosomes or lysosomes in these cells. The results indicate that

  3. Genistein and Glyceollin Effects on ABCC2 (MRP2 and ABCG2 (BCRP in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Chandler Schexnayder

    2015-12-01

    Full Text Available The goal of the present study was to determine the effects of glyceollins on intestinal ABCC2 (ATP Binding Cassette C2, multidrug resistance protein 2, MRP2 and ABCG2 (ATP Binding Cassette G2, breast cancer resistance protein, BCRP function using the Caco-2 cell intestinal epithelial cell model. Glyceollins are soy-derived phytoestrogens that demonstrate anti-proliferative activity in several sources of cancer cells. 5 (and 6-carboxy-2′,7′-dichloroflourescein (CDF was used as a prototypical MRP2 substrate; whereas BODIPY-prazosin provided an indication of BCRP function. Comparison studies were conducted with genistein. Glyceollins were shown to inhibit MRP2-mediated CDF transport, with activity similar to the MRP2 inhibitor, MK-571. They also demonstrated concentration-dependent inhibition BCRP-mediated efflux of BODIPY-prazosin, with a potency similar to that of the recognized BCRP inhibitor, Ko143. In contrast, genistein did not appear to alter MRP2 activity and even provided a modest increase in BCRP efflux of BODIPY-prazosin. In particular, glyceollin inhibition of these two important intestinal efflux transporters suggests the potential for glyceollin to alter the absorption of other phytochemicals with which it might be co-administered as a dietary supplement, as well as alteration of the absorption of pharmaceuticals that may be administered concomitantly.

  4. The Role of Sodium-Dependent Glucose Transporter 1 and Glucose Transporter 2 in the Absorption of Cyanidin-3-O-β-Glucoside in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Tang-Bin Zou

    2014-10-01

    Full Text Available Anthocyanins have multiple biological activities of benefit to human health. While a few studies have been conducted to evaluate the bioavailability of anthocyanins, the mechanisms of their absorption mechanism remain ill-defined. In the present study, we investigated the absorption mechanism of cyanidin-3-O-β-glucoside (Cy-3-G in human intestinal epithelial (Caco-2 cells. Cy-3-G transport was assessed by measuring the absorptive and efflux direction. Inhibition studies were conducted using the pharmacological agents, phloridzin, an inhibitor of sodium-dependent glucose transporter 1 (SGLT1, or phloretin, an inhibitor of glucose transporter 2 (GLUT2. The results showed that phloridzin and phloretin significantly inhibited the absorption of Cy-3-G. In addition, Caco-2 cells transfected with small interfering RNA (siRNA specific for SGLT1 or GLUT2 showed significantly decreased Cy-3-G absorption. These siRNA transfected cells also showed a significantly decreased rate of transport of Cy-3-G compared with the control group. These findings suggest that Cy-3-G absorption is dependent on the activities of SGLT1 and GLUT2 in the small intestine and that SGLT1 and GLUT2 could be a limiting step for the bioavailability of Cy-3-G.

  5. Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.

    Directory of Open Access Journals (Sweden)

    Nan Ye Lei

    Full Text Available Intestinal epithelial stem cells (ISCs are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

  6. Serratia marcescens is injurious to intestinal epithelial cells.

    Science.gov (United States)

    Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

  7. Lipoxin A4 modulates transmigration of human neutrophils across intestinal epithelial monolayers.

    OpenAIRE

    Colgan, S P; Serhan, C N; Parkos, C A; Delp-Archer, C; Madara, J L

    1993-01-01

    Neutrophil (PMN) migration across intestinal epithelial barriers, such as occurs in many disease states, results in modifications in epithelial barrier. Here, we investigated the impact of lipoxin A4 (LXA4), an eicosanoid with counterregulatory inflammatory roles, on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was assessed in the apical-to-basolateral direction and in the basolateral-to-apical direction. In the ap...

  8. Arginine consumption by the intestinal parasite Giardia intestinalis reduces proliferation of intestinal epithelial cells.

    Science.gov (United States)

    Stadelmann, Britta; Merino, María C; Persson, Lo; Svärd, Staffan G

    2012-01-01

    In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the hosts production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that

  9. Arginine consumption by the intestinal parasite Giardia intestinalis reduces proliferation of intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Britta Stadelmann

    Full Text Available In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the hosts production of the antimicrobial agent nitric oxide (NO. A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI. Reduced intestinal epithelial cell (IEC proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful

  10. Rhubarb Supplementation Promotes Intestinal Mucosal Innate Immune Homeostasis through Modulating Intestinal Epithelial Microbiota in Goat Kids.

    Science.gov (United States)

    Jiao, Jinzhen; Wu, Jian; Wang, Min; Zhou, Chuanshe; Zhong, Rongzhen; Tan, Zhiliang

    2018-01-31

    The abuse and misuse of antibiotics in livestock production pose a potential health risk globally. Rhubarb can serve as a potential alternative to antibiotics, and several studies have looked into its anticancer, antitumor, and anti-inflammatory properties. The aim of this study was to test the effects of rhubarb supplementation to the diet of young ruminants on innate immune function and epithelial microbiota in the small intestine. Goat kids were fed with a control diet supplemented with or without rhubarb (1.25% DM) and were slaughtered at days 50 and 60 of age. Results showed that the supplementation of rhubarb increased ileal villus height (P = 0.036), increased jejujal and ileal anti-inflammatory IL-10 production (P innate immune function were accompanied by shifts in ileal epithelial bacterial ecosystem in favor of Blautia, Clostridium, Lactobacillus, and Pseudomonas, and with a decline in the relative abundance of Staphylococcus (P innate immune homeostasis by modulating intestinal epithelial microbiota during the early stages of animal development.

  11. Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.

    Science.gov (United States)

    Zhao, Wei; Li, Xin; Liu, Wen-Hui; Zhao, Jian; Jin, Yi-Ming; Sui, Ting-Ting

    2014-09-01

    Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Activation of intestinal epithelial Stat3 orchestrates tissue defense during gastrointestinal infection.

    Science.gov (United States)

    Wittkopf, Nadine; Pickert, Geethanjali; Billmeier, Ulrike; Mahapatro, Mousumi; Wirtz, Stefan; Martini, Eva; Leppkes, Moritz; Neurath, Markus Friedrich; Becker, Christoph

    2015-01-01

    Gastrointestinal infections with EHEC and EPEC are responsible for outbreaks of diarrheal diseases and represent a global health problem. Innate first-line-defense mechanisms such as production of mucus and antimicrobial peptides by intestinal epithelial cells are of utmost importance for host control of gastrointestinal infections. For the first time, we directly demonstrate a critical role for Stat3 activation in intestinal epithelial cells upon infection of mice with Citrobacter rodentium - a murine pathogen that mimics human infections with attaching and effacing Escherichia coli. C. rodentium induced transcription of IL-6 and IL-22 in gut samples of mice and was associated with activation of the transcription factor Stat3 in intestinal epithelial cells. C. rodentium infection induced expression of several antimicrobial peptides such as RegIIIγ and Pla2g2a in the intestine which was critically dependent on Stat3 activation. Consequently, mice with specific deletion of Stat3 in intestinal epithelial cells showed increased susceptibility to C. rodentium infection as indicated by high bacterial load, severe gut inflammation, pronounced intestinal epithelial cell death and dissemination of bacteria to distant organs. Together, our data implicate an essential role for Stat3 activation in intestinal epithelial cells during C. rodentium infection. Stat3 concerts the host response to bacterial infection by controlling bacterial growth and suppression of apoptosis to maintain intestinal epithelial barrier function.

  13. Activation of intestinal epithelial Stat3 orchestrates tissue defense during gastrointestinal infection.

    Directory of Open Access Journals (Sweden)

    Nadine Wittkopf

    Full Text Available Gastrointestinal infections with EHEC and EPEC are responsible for outbreaks of diarrheal diseases and represent a global health problem. Innate first-line-defense mechanisms such as production of mucus and antimicrobial peptides by intestinal epithelial cells are of utmost importance for host control of gastrointestinal infections. For the first time, we directly demonstrate a critical role for Stat3 activation in intestinal epithelial cells upon infection of mice with Citrobacter rodentium - a murine pathogen that mimics human infections with attaching and effacing Escherichia coli. C. rodentium induced transcription of IL-6 and IL-22 in gut samples of mice and was associated with activation of the transcription factor Stat3 in intestinal epithelial cells. C. rodentium infection induced expression of several antimicrobial peptides such as RegIIIγ and Pla2g2a in the intestine which was critically dependent on Stat3 activation. Consequently, mice with specific deletion of Stat3 in intestinal epithelial cells showed increased susceptibility to C. rodentium infection as indicated by high bacterial load, severe gut inflammation, pronounced intestinal epithelial cell death and dissemination of bacteria to distant organs. Together, our data implicate an essential role for Stat3 activation in intestinal epithelial cells during C. rodentium infection. Stat3 concerts the host response to bacterial infection by controlling bacterial growth and suppression of apoptosis to maintain intestinal epithelial barrier function.

  14. Caco-2 cells - expression, regulation and function of drug transporters compared with human jejunal tissue.

    Science.gov (United States)

    Brück, S; Strohmeier, J; Busch, D; Drozdzik, M; Oswald, S

    2017-03-01

    Induction or inhibition of drug transporting proteins by concomitantly administered drugs can cause serious drug-drug interactions (DDIs). However, in vitro assays currently available are mostly for studying the inhibitory potential of drugs on intestinal transporter proteins, rather than induction. Therefore, this study investigated the suitability of the frequently used intestinal Caco-2 cell line to predict transporter-mediated DDIs as caused by induction via activation of nuclear receptors. TaqMan® low density arrays and LC-MS/MS based targeted proteomics were used to evaluate transporter expression in Caco-2 cells in comparison with jejunal tissue, in culture-time dependence studies and after incubation with different known inducers of drug metabolism and transport. Additionally, studies on ABCB1 function were performed using Transwell® assays with [3 H]-digoxin and [3 H]-talinolol as substrates after incubation with the prototypical inducers rifampicin, St John's wort, carbamazepine and efavirenz. The gene and protein expression pattern of drug transporters in Caco-2 cells and jejunal tissue differed considerably. For some transporters culture-time dependent differences in mRNA expression and/or protein abundance could be determined. Finally, none of the studied prototypical inducers showed an effect either on mRNA expression and protein abundance or on the function of ABCB1. Differences in transporter expression in Caco-2 cells compared with jejunal tissue, as well as expression dependence on culture time must be considered in in vitro studies to avoid under- or overestimation of certain transporters. The Caco-2 cell model is not suitable for the evaluation of DDIs caused by transporter induction. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Mitochondrial ATP Depletion Disrupts Caco-2 Monolayer Integrity and Internalizes Claudin 7.

    Science.gov (United States)

    JanssenDuijghuijsen, Lonneke M; Grefte, Sander; de Boer, Vincent C J; Zeper, Lara; van Dartel, Dorien A M; van der Stelt, Inge; Bekkenkamp-Grovenstein, Melissa; van Norren, Klaske; Wichers, Harry J; Keijer, Jaap

    2017-01-01

    Objective:In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Methods: Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed. Results: Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins (TJP) 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Conclusions: Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the

  16. The Proteome of Filter-Grown Caco-2 Cells With a Focus on Proteins Involved in Drug Disposition.

    Science.gov (United States)

    Ölander, Magnus; Wiśniewski, Jacek R; Matsson, Pär; Lundquist, Patrik; Artursson, Per

    2016-02-01

    Caco-2 cells are widely used in studies of intestinal cell physiology and drug transport. Here, the global proteome of filter-grown Caco-2 cells was quantified using the total protein approach and compared with the human colon and jejunum proteomes. In total, 8096 proteins were identified. In-depth analysis of proteins defining enterocyte differentiation-including brush-border hydrolases, integrins, and adherens and tight junctions-gave near-complete coverage of the expected proteins. Three hundred twenty-seven absorption, distribution, metabolism and excretion proteins were identified, including 112 solute carriers and 20 ATP-binding cassette transporters. OATP2B1 levels were 16-fold higher in Caco-2 cells than in jejunum. To investigate the impact of this difference on in vitro-in vivo extrapolations, we studied the uptake kinetics of the OATP2B1 substrate pitavastatin in Caco-2 monolayers, and found that the contribution of OATP2B1 was 60%-70% at clinically relevant intestinal concentrations. Pitavastatin kinetics was combined with transporter concentrations to model the contribution of active transport and membrane permeation in the jejunum. The lower OATP2B1 expression in jejunum led to a considerably lower transporter contribution (Caco-2 proteome. We also demonstrate the crucial importance of considering transporter expression levels for correct interpretation of drug transport routes across the human intestine. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  17. Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy.

    Science.gov (United States)

    Tang, Yulong; Li, Fengna; Tan, Bie; Liu, Gang; Kong, Xiangfeng; Hardwidge, Philip R; Yin, Yulong

    2014-06-25

    The morbidity and mortality in piglets caused by enterotoxigenic Escherichia coli (ETEC) results in large economic losses to the swine industry, but the precise pathogenesis of ETEC-associated diseases remains unknown. Intestinal epithelial cell autophagy serves as a host defense against pathogens. We found that ETEC induced autophagy, as measured by both the increased punctae distribution of GFP-LC3 and the enhanced conversion of LC3-I to LC3-II. Inhibiting autophagy resulted in decreased survival of IPEC-1 cells infected with ETEC. ETEC triggered autophagy in IPEC-1 cells through a pathway involving the mammalian target of rapamycin (mTOR), the extracellular signal-regulated kinases 1/2 (ERK1/2), and the AMP-activated protein kinase (AMPK). Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Caco-2 cell permeability modelling: a neural network coupled genetic algorithm approach

    Science.gov (United States)

    Di Fenza, Armida; Alagona, Giuliano; Ghio, Caterina; Leonardi, Riccardo; Giolitti, Alessandro; Madami, Andrea

    2007-04-01

    The ability to cross the intestinal cell membrane is a fundamental prerequisite of a drug compound. However, the experimental measurement of such an important property is a costly and highly time consuming step of the drug development process because it is necessary to synthesize the compound first. Therefore, in silico modelling of intestinal absorption, which can be carried out at very early stages of drug design, is an appealing alternative procedure which is based mainly on multivariate statistical analysis such as partial least squares (PLS) and neural networks (NN). Our implementation of neural network models for the prediction of intestinal absorption is based on the correlation of Caco-2 cell apparent permeability ( P app) values, as a measure of intestinal absorption, to the structures of two different data sets of drug candidates. Several molecular descriptors of the compounds were calculated and the optimal subsets were selected using a genetic algorithm; therefore, the method was indicated as Genetic Algorithm-Neural Network (GA-NN). A methodology combining a genetic algorithm search with neural network analysis applied to the modelling of Caco-2 P app has never been presented before, although the two procedures have been already employed separately. Moreover, we provide new Caco-2 cell permeability measurements for more than two hundred compounds. Interestingly, the selected descriptors show to possess physico-chemical connotations which are in excellent accordance with the well known relevant molecular properties involved in the cellular membrane permeation phenomenon: hydrophilicity, hydrogen bonding propensity, hydrophobicity and molecular size. The predictive ability of the models, although rather good for a preliminary study, is somewhat affected by the poor precision of the experimental Caco-2 measurements. Finally, the generalization ability of one model was checked on an external test set not derived from the data sets used to build the models

  19. Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Sourav, E-mail: sourav.bhattacharjee@wur.nl [Wageningen University, Laboratory of Organic Chemistry (Netherlands); Opstal, Edward J. van; Alink, Gerrit M. [Wageningen University, Division of Toxicology (Netherlands); Marcelis, Antonius T. M.; Zuilhof, Han [Wageningen University, Laboratory of Organic Chemistry (Netherlands); Rietjens, Ivonne M. C. M. [Wageningen University, Division of Toxicology (Netherlands)

    2013-06-15

    The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size {approx}45 nm) and polystyrene nanoparticles (PSNPs/size {approx}50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

  20. Application of Caco-2 Cell Line in Herb-Drug Interaction Studies: Current Approaches and Challenges

    Science.gov (United States)

    Awortwe, C.; Fasinu, P.S.; Rosenkranz, B.

    2015-01-01

    The Caco-2 model is employed in pre-clinical investigations to predict the likely gastrointestinal permeability of drugs because it expresses cytochrome P450 enzymes, transporters, microvilli and enterocytes of identical characteristics to the human small intestine. The FDA recommends this model as integral component of the Biopharmaceutics Classification System (BCS). Most dedicated laboratories use the Caco-2 cell line to screen new chemical entities through prediction of its solubility, bioavailability and the possibility of drug-drug or herb-drug interactions in the gut lumen. However, challenges in the inherent characteristics of Caco-2 cell and inter-laboratory protocol variations have resulted to generation of irreproducible data. These limitations affect the extrapolation of data from pre-clinical research to clinical studies involving drug-drug and herb-drug interactions. This review addresses some of these caveats and enumerates the plausible current and future approaches to reduce the anomalies associated with Caco-2 cell line investigations focusing on its application in herb-drug interactions. PMID:24735758

  1. Formation of a Hydroxymethylfurfural-Cysteine Adduct and Its Absorption and Cytotoxicity in Caco-2 Cells.

    Science.gov (United States)

    Zhao, Qianzhu; Zou, Yueyu; Huang, Caihuan; Lan, Ping; Zheng, Jie; Ou, Shiyi

    2017-11-15

    Adducts of 5-hydroxymethylfurfural (HMF)-amino acids are formed during food processing and digestion; the elimination capacity of in vitro intestinal digests of biscuits, instant noodles, and potato crisps for HMF is 652, 727, and 540 μg/g, respectively. However, the safety of these adducts is unknown. In this study, an HMF-cysteine adduct named 1-dicysteinethioacetal-5-hydroxymehtylfurfural (DCH), which was found to be produced in the gastrointestinal tract after HMF intake, was prepared to test its effect toward Caco-2 cells. Compared with HMF, the adduct displayed lower cytotoxicity against Caco-2 cells with an IC50 value of 31.26 mM versus 14.95 mM (HMF). The DCH did not induce cell apoptosis, whereas HMF significantly increased the apoptosis rate after incubation at concentrations of 16, 32, and 48 mM for 72 h. DCH showed an absorption rate considerably lower than that of HMF by Caco-2 cells. Lower absorption of DCH may result in lower toxicity compared with HMF against Caco-2 cells. Intracellular transformation of DCH has been observed.

  2. Transgenic Expression of Human Lysophosphatidic Acid Receptor LPA2 in Mouse Intestinal Epithelial Cells Induces Intestinal Dysplasia.

    Directory of Open Access Journals (Sweden)

    Michihiro Yoshida

    Full Text Available Lysophosphatidic acid (LPA acts on LPA2 receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA2 attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA2 alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA2 in intestinal epithelial cells (IECs under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG human LPA2; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA2 colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA2 compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA2 overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA2 alone can lead to intestinal dysplasia.

  3. Colostrum whey down-regulates the expression of early and late inflammatory response genes induced by Escherichia coli and Salmonella enterica Typhimurium components in intestinal epithelial cells.

    Science.gov (United States)

    Blais, M; Fortier, M; Pouliot, Y; Gauthier, S F; Boutin, Y; Asselin, C; Lessard, M

    2015-01-28

    Pathogenic invasion by Escherichia coli and Salmonellae remains a constant threat to the integrity of the intestinal epithelium and can rapidly induce inflammatory responses. At birth, colostrum consumption exerts numerous beneficial effects on the properties of intestinal epithelial cells and protects the gastrointestinal tract of newborns from pathogenic invasion. The present study aimed to investigate the effect of colostrum on the early and late inflammatory responses induced by pathogens. The short-term (2 h) and long-term (24 h) effects of exposure to heat-killed (HK) E. coli and Salmonella enterica Typhimurium on gene expression in the porcine intestinal epithelial cell (IPEC-J2) model were first evaluated by microarray and quantitative PCR analyses. Luciferase assays were performed using a NF-κB-luc reporter construct to investigate the effect of colostrum whey treatment on the activation of NF-κB induced by HK bacteria. Luciferase assays were also performed using NF-κB-luc, IL-8-luc and IL-6-luc reporter constructs in human colon adenocarcinoma Caco-2/15 cells exposed to dose-response stimulations with HK bacteria and colostrum whey. Bovine colostrum whey treatment decreased the expression of early and late inflammatory genes induced by HK bacteria in IPEC-J2, as well as the transcriptional activation of NF-κB-luc induced by HK bacteria. Unlike that with colostrum whey, treatment with other milk fractions failed to decrease the activation of NF-κB-luc induced by HK bacteria. Lastly, the reduction of the HK bacteria-induced activation of NF-κB-luc, IL-8-luc and IL-6-luc by colostrum whey was dose dependent. The results of the present study indicate that bovine colostrum may protect and preserve the integrity of the intestinal mucosal barrier in the host by controlling the expression levels of early and late inflammatory genes following invasion by enteric pathogens.

  4. Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung-Min [Department of Biological Sciences, University of Columbia, NY (United States); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Attieh, Zouhair K. [Department of Laboratory Science and Technology, American University of Science and Technology, Ashrafieh (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Son, Hee Sook [Department of Food Science and Human Nutrition, College of Human Ecology, Chonbuk National University (Korea, Republic of); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Chen, Huijun [Medical School, Nanjing University, Nanjing 210008, Jiangsu Province (China); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Bacouri-Haidar, Mhenia [Department of Biology, Faculty of Sciences (I), Lebanese University, Hadath (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Vulpe, Chris D., E-mail: vulpe@berkeley.edu [Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in non-polarized cells. Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in iron deficient and polarized cells. Black-Right-Pointing-Pointer Hephaestin with apical iron moves near to basolateral membrane of polarized cells. Black-Right-Pointing-Pointer Peri-basolateral location of hephaestin is accessible to the extracellular space. Black-Right-Pointing-Pointer Hephaestin is involved in iron mobilization from the intestine to circulation. -- Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO{sub 4}) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a

  5. Epithelial CaSR Deficiency Alters Intestinal Integrity and Promotes Proinflammatory Immune Responses

    OpenAIRE

    Cheng, Sam X.; Lightfoot, Yaíma L.; Yang, Tao; Zadeh, Mojgan; Tang, Lieqi; Sahay, Bikash; Wang, Gary P.; Owen, Jennifer L.; Mohamadzadeh, Mansour

    2014-01-01

    The intestinal epithelium is equipped with sensing receptor mechanisms that interact with luminal microorganisms and nutrients to regulate barrier function and gut immune responses, thereby maintaining intestinal homeostasis. Herein, we clarify the role of the extracellular calcium-sensing receptor (CaSR) using intestinal epithelium-specific Casr−/− mice. Epithelial CaSR deficiency diminished intestinal barrier function, altered microbiota composition, and skewed immune responses towards proi...

  6. Titanium Dioxide Particle Type and Concentration Influence the Inflammatory Response in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Saeko Tada-Oikawa

    2016-04-01

    Full Text Available Titanium dioxide (TiO2 nanoparticles are widely used in cosmetics, sunscreens, biomedicine, and food products. When used as a food additive, TiO2 nanoparticles are used in significant amounts as white food-coloring agents. However, the effects of TiO2 nanoparticles on the gastrointestinal tract remain unclear. The present study was designed to determine the effects of five TiO2 particles of different crystal structures and sizes in human epithelial colorectal adenocarcinoma (Caco-2 cells and THP-1 monocyte-derived macrophages. Twenty-four-hour exposure to anatase (primary particle size: 50 and 100 nm and rutile (50 nm TiO2 particles reduced cellular viability in a dose-dependent manner in THP-1 macrophages, but in not Caco-2 cells. However, 72-h exposure of Caco-2 cells to anatase (50 nm TiO2 particles reduced cellular viability in a dose-dependent manner. The highest dose (50 µg/mL of anatase (100 nm, rutile (50 nm, and P25 TiO2 particles also reduced cellular viability in Caco-2 cells. The production of reactive oxygen species tended to increase in both types of cells, irrespective of the type of TiO2 particle. Exposure of THP-1 macrophages to 50 µg/mL of anatase (50 nm TiO2 particles increased interleukin (IL-1β expression level, and exposure of Caco-2 cells to 50 µg/mL of anatase (50 nm TiO2 particles also increased IL-8 expression. The results indicated that anatase TiO2 nanoparticles induced inflammatory responses compared with other TiO2 particles. Further studies are required to determine the in vivo relevance of these findings to avoid the hazards of ingested particles.

  7. Pseudomonas aeruginosa serA Gene Is Required for Bacterial Translocation through Caco-2 Cell Monolayers.

    Science.gov (United States)

    Yasuda, Masashi; Nagata, Syouya; Yamane, Satoshi; Kunikata, Chinami; Kida, Yutaka; Kuwano, Koichi; Suezawa, Chigusa; Okuda, Jun

    2017-01-01

    To specify critical factors responsible for Pseudomonas aeruginosa penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to P. aeruginosa PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the serA mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the serA gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type P. aeruginosa PAO1 strain. Together, these results suggest that serA is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Oral administration of L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by P. aeruginosa through inhibition of serA function.

  8. Effects of nonsteroidal anti-inflammatory drugs on the expression and function of P-glycoprotein/MDR1 in Caco-2 cells.

    Science.gov (United States)

    Takara, Kohji; Hayashi, Ryuhei; Kokufu, Misato; Yamamoto, Kazuhiro; Kitada, Noriaki; Ohnishi, Noriaki; Yokoyama, Teruyoshi

    2009-01-01

    The aim of this study was to examine the effects of 16 kinds of nonsteroidal anti-inflammatory drugs (NSAIDs) on P-glycoprotein/MDR1 in Caco-2 cells as an intestinal epithelial cell model. Cells were treated with NSAIDs for 24 hours, and then, the expression of MDR1 mRNA was evaluated by reverse-transcriptase polymerase chain reaction. The function of MDR1 in cells pretreated with NSAIDs for 48 hours was evaluated by measuring the cellular amount of rhodamine123, which is a substrate of MDR1. The expression of MDR1 mRNA was increased by diclofenac, fenbufen, indomethacin, and nimesulide and the tended to be increased by meloxicam, mepirizole, and sulindac. However, pretreatment for 48 hours with diclofenac, indomethacin, or nimesulide, but not fenbufen, resulted in a significant increase in the amount of rhodamine123 accumulated. Although NSAIDs without effects on the expression of MDR1 mRNA altered the accumulation of rhodamine123 significantly, the efflux of rhodamine123 from cells was unchanged. In conclusion, the expression of MDR1 mRNA in Caco-2 cells was demonstrated to be increased by treatment with some NSAIDs, although the transport function of MDR1 was unchanged. These findings imply that the NSAIDs did not cause the drug interaction via MDR1 induction.

  9. Antagonistics against pathogenic Bacillus cereus in milk fermentation by Lactobacillus plantarum ZDY2013 and its anti-adhesion effect on Caco-2 cells against pathogens.

    Science.gov (United States)

    Zhang, Zhihong; Tao, Xueying; Shah, Nagendra P; Wei, Hua

    2016-04-01

    Lactobacillus plantarum ZDY2013 is a potential probiotic isolated from fermented bean acid. In this study, we aimed to evaluate the in vitro antimicrobial activity of this organism against Bacillus cereus in milk fermentation, the antiadhesion ability on intestinal epithelial cells, as well as its ability to abrogate the cytotoxic effect and expression levels of genes. We found no antimicrobial activity produced by L. plantarum once the pH was adjusted to 6.0 and 7.0. The pH decreased continuously when L. plantarum and B. cereus were co-incubated during milk fermentation, which caused a decrease in the B. cereus counts. Antiadhesion assays showed that L. plantarum can significantly inhibit the adhesion of enterotoxin-producing B. cereus ATCC14579 and pathogenic B. cereus HN001 by inhibition, competition, and displacement. The supernatants of B. cereus, either alone or in conjunction with L. plantarum, caused damage to the membrane integrity of Caco-2 cells to release lactate dehydrogenase. In addition, L. plantarum tended to attenuate proinflammatory cytokine and oxidative stress gene expression on Caco-2 cells, inducing with B. cereus HN001 supernatants. This study provided systematic insights into the antagonistic effect of L. plantarum ZDY2013, and the information may be helpful to explore potential control measures for preventing food poisoning by lactic acid bacteria. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. Krüppel-like factor 4 regulates intestinal epithelial cell morphology and polarity.

    Directory of Open Access Journals (Sweden)

    Tianxin Yu

    Full Text Available Krüppel-like factor 4 (KLF4 is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT, was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell

  11. Low-molecular-weight fucoidan and high-stability fucoxanthin from brown seaweed exert prebiotics and anti-inflammatory activities in Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Pai-An Hwang

    2016-08-01

    Full Text Available Background: The aim of this study is to investigate the anti-inflammatory effects of low-molecular-weight fucoidan (LMF and high-stability fucoxanthin (HS-Fucox in a lipopolysaccharide-induced inflammatory Caco-2 cell line co-culture with B. lactis. Methods: We used various methods such as transepithelial resistance (TER assay, cytokine secretion assay, and tight junction protein mRNA expression assay to examine LMF and HS-Fucox anti-inflammatory properties. Results: LMF and HS-Fucox activated probiotic growth and reduced the inflammation of the intestinal epithelial cells. Moreover, the combination of LMFHS-Fucox dramatically enhanced the intestinal epithelial barrier and immune function against the lipopolysaccharide effect by inhibiting IL-1β and TNF-α and promoting IL-10 and IFN-γ. Conclusion: These findings suggested that LMF and HS-Fucox, alone or in combination, could be the potential natural compounds to enhance the immune system and have an anti-inflammatory effect on the intestinal cells.

  12. Comparative evaluation of nano-CuO crossing Caco-2 cell monolayers and cellular uptake

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Gao; Lianqin, Zhu, E-mail: lianqinz1963@163.com; Fenghua, Zhu [Qingdao Agricultural University, College of Animal Science and Veterinary Medicine (China); Fang, Zheng [Dezhou University, College of Agriculture (China); Mingming, Song; Kai, Huang [Qingdao Agricultural University, College of Animal Science and Veterinary Medicine (China)

    2015-04-15

    Different concentrations of CuSO{sub 4}, micro-CuO, and nano-CuO were added to Caco-2 cell monolayers to study the absorption and transport characteristics in this epithelial cell model. Nano-CuO nanoparticles had a diameter of 10–20 nm. Inhibitors of endocytosis were used to explore whether nano-CuO could enter the Caco-2 cell in the form of nanoparticles, and to ascertain the endocytotic pathway that is involved in the transport process. The apparent permeability coefficient (P{sub app}) of CuSO{sub 4} and nano-CuO increased with the Cu concentration in the culture medium (p < 0.05). The micro-CuO of different concentrations had no significant impact on the P{sub app} value of Caco-2 cells (p > 0.05). When the Cu concentration in the culture medium was in the range 31.25–500 μM, the P{sub app} value of Caco-2 cells incubated with nano-CuO was significantly higher than that obtained with CuSO{sub 4}. The latter was also significantly higher than that when cells were incubated with micro-CuO (p < 0.05). The amount of Cu transport increased with the increase of CuSO{sub 4} concentration in the culture medium. After 90 min, the amount of transport began to saturate, and the transport rate of Cu declined with the increase of CuSO{sub 4} concentration. For the cells incubated with nano-CuO, the amount of Cu transport increased with the increase of nano-CuO concentration, but did not show an obvious saturation with the extension of transport time. Nano-CuO could enter the Caco-2 cell in the form of nanoparticles, and were found in the cytoplasm, vesicles, lysosomes, and cell nuclei. Several inhibitors of endocytosis effectively prevented the entry of nano-CuO into the Caco-2 cells. It was concluded that nano-CuO particles can enter the Caco-2 cells through several cellular endocytotic pathways.

  13. Transepithelial Transport of Curcumin in Caco-2 Cells Is significantly Enhanced by Micellar Solubilisation.

    Science.gov (United States)

    Frank, Jan; Schiborr, Christina; Kocher, Alexa; Meins, Jürgen; Behnam, Dariush; Schubert-Zsilavecz, Manfred; Abdel-Tawab, Mona

    2017-03-01

    Curcumin, the active constituent of Curcuma longa L. (family Zingiberaceae), has gained increasing interest because of its anti-cancer, anti-inflammatory, anti-diabetic, and anti-rheumatic properties associated with good tolerability and safety up to very high doses of 12 g. Nanoscaled micellar formulations on the base of Tween 80 represent a promising strategy to overcome its low oral bioavailability. We therefore aimed to investigate the uptake and transepithelial transport of native curcumin (CUR) vs. a nanoscaled micellar formulation (Sol-CUR) in a Caco-2 cell model. Sol-CUR afforded a higher flux than CUR (39.23 vs. 4.98 μg min(-1) cm(-2), respectively). This resulted in a higher Papp value of 2.11 × 10(-6) cm/s for Sol-CUR compared to a Papp value of 0.56 × 10(-6) cm/s for CUR. Accordingly a nearly 9.5 fold higher amount of curcumin was detected on the basolateral side at the end of the transport experiments after 180 min with Sol-CUR compared to CUR. The determined 3.8-fold improvement in the permeability of curcumin is in agreement with an up to 185-fold increase in the AUC of curcumin observed in humans following the oral administration of the nanoscaled micellar formulation compared to native curcumin. The present study demonstrates that the enhanced oral bioavailability of micellar curcumin formulations is likely a result of enhanced absorption into and increased transport through small intestinal epithelial cells.

  14. Simulating kinetic parameters in transporter mediated permeability across Caco-2 cells. A case study on estrange-3-sulphate

    DEFF Research Database (Denmark)

    Rolsted, Kamilla; Rapin, Nicolas; Steffansen, Bente

    2011-01-01

    Substances that compete for the same saturable intestinal transporters may when dosed together lead to altered permeability and hence influence bioavailability. The aim was to simulate kinetic parameters, i.e. K(m) and J(max), for transporter mediated E(1)S permeability across Caco-2 cells...

  15. Strain-Dependent Induction of Human Enterocyte Apoptosis by Blastocystis Disrupts Epithelial Barrier and ZO-1 Organization in a Caspase 3- and 9-Dependent Manner

    Science.gov (United States)

    Teo, Joshua D. W.; Tan, Kevin S. W.

    2014-01-01

    Blastocystis is an emerging protistan parasite colonizing the human intestine. It is frequently reported to cause general intestinal symptoms of vomiting, diarrhea, and abdominal pain. We recently demonstrated that Blastocystis rearranged cytoskeletal proteins and induced intestinal epithelial barrier compromise. The effect of Blastocystis on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and increased epithelial permeability has yet to be determined. The aim of this study is to assess if Blastocystis induces human enterocyte apoptosis and whether this effect influences human intestinal epithelial barrier function. Monolayers of polarized human colonic epithelial cell-line Caco-2 were incubated with Blastocystis subtype 7 and subtype 4. Assays for both early and late markers of apoptosis, phosphatidylserine externalization, and nuclear fragmentation, respectively, showed that Blastocystis ST-7, but not ST-4, significantly increased apoptosis in enterocytes, suggesting that Blastocystis exhibits host specificity and strain-to-strain variation in pathogenicity. ST-7 also activated Caco-2 caspases 3 and 9 but not 8. ST-7 induced changes in epithelial resistance, permeability, and tight junction (ZO-1) localization. Pretreatment of Caco-2 monolayers with a pan-caspase inhibitor z-VAD-fmk significantly inhibited these changes. This suggests a role for enterocyte apoptosis in Blastocystis-mediated epithelial barrier compromise in the human intestine. PMID:24822183

  16. Strain-Dependent Induction of Human Enterocyte Apoptosis by Blastocystis Disrupts Epithelial Barrier and ZO-1 Organization in a Caspase 3- and 9-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Zhaona Wu

    2014-01-01

    Full Text Available Blastocystis is an emerging protistan parasite colonizing the human intestine. It is frequently reported to cause general intestinal symptoms of vomiting, diarrhea, and abdominal pain. We recently demonstrated that Blastocystis rearranged cytoskeletal proteins and induced intestinal epithelial barrier compromise. The effect of Blastocystis on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and increased epithelial permeability has yet to be determined. The aim of this study is to assess if Blastocystis induces human enterocyte apoptosis and whether this effect influences human intestinal epithelial barrier function. Monolayers of polarized human colonic epithelial cell-line Caco-2 were incubated with Blastocystis subtype 7 and subtype 4. Assays for both early and late markers of apoptosis, phosphatidylserine externalization, and nuclear fragmentation, respectively, showed that Blastocystis ST-7, but not ST-4, significantly increased apoptosis in enterocytes, suggesting that Blastocystis exhibits host specificity and strain-to-strain variation in pathogenicity. ST-7 also activated Caco-2 caspases 3 and 9 but not 8. ST-7 induced changes in epithelial resistance, permeability, and tight junction (ZO-1 localization. Pretreatment of Caco-2 monolayers with a pan-caspase inhibitor z-VAD-fmk significantly inhibited these changes. This suggests a role for enterocyte apoptosis in Blastocystis-mediated epithelial barrier compromise in the human intestine.

  17. Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform

    Directory of Open Access Journals (Sweden)

    Kenji Kozuka

    2017-12-01

    Full Text Available Summary: We describe the development and characterization of a mouse and human epithelial cell monolayer platform of the small and large intestines, with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages. Ion transport phenotypes of monolayers from the proximal and distal colon and small intestine matched the known and unique physiology of these intestinal segments. The cultures secreted serotonin, GLP-1, and FGF19 and upregulated the epithelial sodium channel in response to known biologically active agents, suggesting intact secretory and absorptive functions. A screen of over 2,000 pharmacologically active compounds for inhibition of potassium ion transport in the mouse distal colon cultures led to the identification of a tool compound. : Siegel and colleagues describe their development of a human and mouse intestinal epithelial cell monolayer platform that maintains the cellular, molecular, and functional characteristics of tissue for each intestinal segment. They demonstrate the platform's application to drug discovery by screening a library of over 2,000 compounds to identify an inhibitor of potassium ion transport in the mouse distal colon. Keywords: intestinal epithelium, organoids, monolayer, colon, small intestine, phenotype screening assays, enteroid, colonoid

  18. Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Kateřina Lněničková

    2016-09-01

    Full Text Available The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE and epigallocatechin gallate (EGCG using both proliferating (P and differentiated (D caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes’ activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well.

  19. Isoflavones in food supplements: chemical profile, label accordance and permeability study in Caco-2 cells.

    Science.gov (United States)

    Almeida, I M C; Rodrigues, F; Sarmento, B; Alves, R C; Oliveira, M B P P

    2015-03-01

    Consumers nowadays are playing an active role in their health-care. A special case is the increasing number of women, who are reluctant to use exogenous hormone therapy for the treatment of menopausal symptoms and are looking for complementary therapies. However, food supplements are not clearly regulated in Europe. The EFSA has only recently begun to address the issues of botanical safety and purity regulation, leading to a variability of content, standardization, dosage, and purity of available products. In this study, isoflavones (puerarin, daidzin, genistin, daidzein, glycitein, genistein, formononetin, prunetin, and biochanin A) from food supplements (n = 15) for menopausal symptoms relief are evaluated and compared with the labelled information. Only four supplements complied with the recommendations made by the EC on the tolerable thresholds. The intestinal bioavailability of these compounds was investigated using Caco-2 cells. The apparent permeability coefficients of the selected isoflavonoids across the Caco-2 cells were affected by the isoflavone concentration and product matrix.

  20. Enteric glial cells and their role in the intestinal epithelial barrier.

    Science.gov (United States)

    Yu, Yan-Bo; Li, Yan-Qing

    2014-08-28

    The intestinal epithelium constitutes a physical and functional barrier between the external environment and the host organism. It is formed by a continuous monolayer of intestinal epithelial cells maintained together by intercellular junctional complex, limiting access of pathogens, toxins and xenobiotics to host tissues. Once this barrier integrity is disrupted, inflammatory disorders and tissue injury are initiated and perpetuated. Beneath the intestinal epithelial cells lies a population of astrocyte-like cells that are known as enteric glia. The morphological characteristics and expression markers of these enteric glia cells were identical to the astrocytes of the central nervous system. In the past few years, enteric glia have been demonstrated to have a trophic and supporting relationship with intestinal epithelial cells. Enteric glia lesions and/or functional defects can be involved in the barrier dysfunction. Besides, factors secreted by enteric glia are important for the regulation of gut barrier function. Moreover, enteric glia have an important impact on epithelial cell transcriptome and induce a shift in epithelial cell phenotype towards increased cell adhesion and cell differentiation. Enteric glia can also preserve epithelial barrier against intestinal bacteria insult. In this review, we will describe the current body of evidence supporting functional roles of enteric glia on intestinal barrier.

  1. Effects of Weaning on Intestinal Upper Villus Epithelial Cells of Piglets

    Science.gov (United States)

    Wang, Xiaocheng; Tan, Bie; Li, Tiejun; Yin, Yulong

    2016-01-01

    The intestinal upper villus epithelial cells represent the differentiated epithelial cells and play key role in digesting and absorbing lumenal nutrients. Weaning stress commonly results in a decrease in villus height and intestinal dysfunction in piglets. However, no study have been conducted to test the effects of weaning on the physiology and functions of upper villus epithelial cells. A total of 40 piglets from 8 litters were weaned at 14 days of age and one piglet from each litter was killed at 0 d (w0d), 1 d (w1d), 3 d (w3d), 5 d (w5d), and 7 d (w7d) after weaning, respectively. The upper villus epithelial cells in mid-jejunum were isolated using the distended intestinal sac method. The expression of proteins in upper villus epithelial cells was analyzed using the isobaric tags for relative and absolute quantification or Western blotting. The expression of proteins involved in energy metabolism, Golgi vesicle transport, protein amino acid glycosylation, secretion by cell, transmembrane transport, ion transport, nucleotide catabolic process, translational initiation, and epithelial cell differentiation and apoptosis, was mainly reduced during the post-weaning period, and these processes may be regulated by mTOR signaling pathway. These results indicated that weaning inhibited various cellular processes in jejunal upper villus epithelial cells, and provided potential new directions for exploring the effects of weaning on the functions of intestine and improving intestinal functions in weaning piglets. PMID:27022727

  2. TLR2 mediates gap junctional intercellular communication through connexin-43 in intestinal epithelial barrier injury.

    Science.gov (United States)

    Ey, Birgit; Eyking, Annette; Gerken, Guido; Podolsky, Daniel K; Cario, Elke

    2009-08-14

    Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

  3. Impact of lipid-based drug delivery systems on the transport and uptake of insulin across Caco-2 Cell monolayers

    DEFF Research Database (Denmark)

    Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

    2016-01-01

    on the transport and uptake mechanisms of insulin across the intestinal membrane. For this purpose, 3 SNEDDS were prepared, and Caco-2 cell monolayers were used to study transport and uptake. The prepared SNEDDSs were all in the range of 35-50 nm and had a negative zeta potential (between -8 and -25 m...... space of the Caco-2 cell monolayer, indicating transport by paracellular diffusion. In contrast, the fluorescein isothiocyanate-labeled insulin in LCT SNEDDS was taken up by the cells. In conclusion, the present study demonstrated that MCT1 and MCT2 SNEDDS, but not LCT SNEDDS increased...

  4. The absorptive flux of the anti-epileptic drug substance vigabatrin is carrier-mediated across Caco-2 cell monolayers

    DEFF Research Database (Denmark)

    Nøhr, Martha Kampp; Hansen, Steen Honoré; Brodin, Birger

    2014-01-01

    of vigabatrin in Caco-2 cells, a cell culture model of the small intestinal epithelium. The uptake and transepithelial flux of vigabatrin was measured using an LC-MS method for quantification. Transepithelial transport of vigabatrin was shown to be proton-dependent and polarized in the apical-to-basolateral (A...... of the human proton-coupled amino acid transporter (hPAT1) to the apical solution. The present study indicates that the transepithelial A-B flux of vigabatrin is mainly mediated by hPAT1 in Caco-2 cells at dose-relevant concentrations....

  5. Epithelial CaSR Deficiency Alters Intestinal Integrity and Promotes Proinflammatory Immune Responses

    Science.gov (United States)

    Cheng, Sam X.; Lightfoot, Yaíma L.; Yang, Tao; Zadeh, Mojgan; Tang, Lieqi; Sahay, Bikash; Wang, Gary P.; Owen, Jennifer L.; Mohamadzadeh, Mansour

    2014-01-01

    The intestinal epithelium is equipped with sensing receptor mechanisms that interact with luminal microorganisms and nutrients to regulate barrier function and gut immune responses, thereby maintaining intestinal homeostasis. Herein, we clarify the role of the extracellular calcium-sensing receptor (CaSR) using intestinal epithelium-specific Casr−/− mice. Epithelial CaSR deficiency diminished intestinal barrier function, altered microbiota composition, and skewed immune responses towards proinflammatory. Consequently, Casr−/− mice were significantly more prone to chemically induced intestinal inflammation resulting in colitis. Accordingly, CaSR represents a potential therapeutic target for autoinflammatory disorders, including inflammatory bowel diseases. PMID:24842610

  6. Peroxisomes in intestinal and gallbladder epithelial cells of the stickleback, Gasterosteus aculeatus L. (Teleostei)

    NARCIS (Netherlands)

    Ruiter, A.J.H. de; Veenhuis, M.; Wendelaar Bonga, S.E.

    1988-01-01

    The occurrence of microbodies in the epithelial cells of the intestine and gallbladder of the stickleback, Gasterosteus aculeatus L., is described. In the intestine the organelles are predominantly located in the apical and perinuclear zone of the cells and may contain small crystalline cores. In

  7. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis

    NARCIS (Netherlands)

    de Jong, Joan H.; Rodermond, Hans M.; Zimberlin, Cheryl D.; Lascano, Valeria; de Sousa E Melo, Felipe; Richel, Dick J.; Medema, Jan Paul; Vermeulen, Louis

    2012-01-01

    The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing

  8. Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

    DEFF Research Database (Denmark)

    López-Posadas, Rocío; Becker, Christoph; Günther, Claudia

    2016-01-01

    Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing t...

  9. Effect of chronic cold stress on intestinal epithelial cell proliferation and inflammation in rats.

    Science.gov (United States)

    Kaushik, Susmita; Kaur, Jyotdeep

    2005-09-01

    The present study evaluated the effect of chronic cold stress on intestinal epithelial cell proliferation and inflammation. Male Wistar rats were subjected to cold exposure for three weeks. At the end of the cold exposure, intestinal cell proliferation, luminal nitrite and protein levels, intestinal myeloperoxidase activity and mast cell numbers were evaluated. Severely compromised proliferation rate of the crypt-base cells was observed under chronic stress conditions. Cells isolated from stressed rats showed a decreased DNA content in villus and lower villus cell fractions and an increased DNA content in the crypt cells, as compared to controls. Chronic cold stress resulted in increased luminal nitrite, luminal protein levels, and intestinal myeloperoxidase activity. The number of mast cells was significantly elevated under chronic stress conditions. Chronic cold stress resulted in a compromised intestinal epithelial cell proliferation rate and induced inflammation in the rat small intestine, through the combined action of nitric oxide, neutrophils and mast cells.

  10. Comparison of the permeability of metoprolol and labetalol in rat, mouse, and Caco-2 cells: use as a reference standard for BCS classification.

    Science.gov (United States)

    Incecayir, Tuba; Tsume, Yasuhiro; Amidon, Gordon L

    2013-03-04

    The purpose of this study was to investigate labetalol as a potential high permeability reference standard for the application of Biopharmaceutics Classification Systems (BCS). Permeabilities of labetalol and metoprolol were investigated in animal intestinal perfusion models and Caco-2 cell monolayers. After isolating specific intestinal segments, in situ single-pass intestinal perfusions (SPIP) were performed in rats and mice. The effective permeabilities (Peff) of labetalol and metoprolol, an FDA standard for the low/high Peff class boundary, were investigated in two different segments of rat intestine (proximal jejunum and distal ileum) and in the proximal jejunum of mouse. No significant difference was found between Peff of metoprolol and labetalol in the jejunum and ileum of rat (0.33 ± 0.11 × 10(-4) vs 0.38 ± 0.06 × 10(-4) and 0.57 ± 0.17 × 10(-4) vs 0.64 ± 0.30 × 10(-4) cm/s, respectively) and in the jejunum of mouse (0.55 ± 0.05 × 10(-4) vs 0.59 ± 0.13 × 10(-4) cm/s). However, Peff of metoprolol and labetalol were 1.7 and 1.6 times higher in the jejunum of mouse, compared to the jejunum of rat, respectively. Metoprolol and labetalol showed segmental-dependent permeability through the rat intestine, with increased Peff in the distal ileum in comparison to the proximal jejunum. Most significantly, Peff of labetalol was found to be concentration-dependent. Decreasing concentrations of labetalol in the perfusate resulted in decreased Peff compared to Peff of metoprolol. The intestinal epithelial permeability of labetalol was lower than that of metoprolol in Caco-2 cells at both apical pH 6.5 and 7.5 (5.96 ± 1.96 × 10(-6) vs 9.44 ± 3.44 × 10(-6) and 15.9 ± 2.2 × 10(-6) vs 23.2 ± 7.1 × 10(-6) cm/s, respectively). Labetalol exhibited higher permeability in basolateral to apical (BL-AP) compared to AP-BL direction in Caco-2 cells at 0.1 times the highest dose strength (HDS) (46.7 ± 6.5 × 10(-6) vs 14.2 ± 1.5 × 10(-6) cm/s). The P

  11. Listeria monocytogenes efficiently invades caco-2 cells after low-temperature storage in broth and on deli meat

    DEFF Research Database (Denmark)

    Larsen, Marianne Halberg; Koch, Anette Granly; Ingmer, Hanne

    2010-01-01

    The objective of this study was to investigate how various growth conditions influence the virulence of Listeria monocytogenes monitored by its ability to invade the epithelial cell lines Caco-2 and INT-407. The growth conditions examined were modified atmosphere-packaged deli meat and brain heart...... infusion broth (BHI) with and without salt. Five strains of L. monocytogenes were selected to investigate their invasiveness and all strains invaded Caco-2 cells at higher levels than INT-407 cells. Further, the clinical strains (3443 and 3734) were more invasive (p ... to invade Caco-2 cells was compared after growth on a fermented sausage and on cured cooked ham to that of bacteria grown in BHI broth supplemented with salt. Samples were stored under chilling conditions for up to 4 weeks. The results showed no difference (p > 0.05) in invasiveness after 7 days at 10...

  12. Effect of Apple, Baobab, Red-Chicory, and Pear Extracts on Cellular Energy Expenditure and Morphology of Caco-2 Cells using Transepithelial Electrical Resistance (TEER) and Scanning Electron Microscopy (SEM)

    Science.gov (United States)

    The present study investigated the effects of four food extracts on the Caco-2 intestinal cell line using a new transepithelial electrical resistance method (TEER) concurrent with electron microscopy (SEM). Caco-2 cells are widely used in transepithelial studies because they can be cultured to creat...

  13. Probiotic-derived polyphosphate enhances the epithelial barrier function and maintains intestinal homeostasis through integrin-p38 MAPK pathway.

    Directory of Open Access Journals (Sweden)

    Shuichi Segawa

    Full Text Available Probiotics exhibit beneficial effects on human health, particularly in the maintenance of intestinal homeostasis in a complex manner notwithstanding the diversity of an intestinal flora between individuals. Thus, it is highly probable that some common molecules secreted by probiotic and/or commensal bacteria contribute to the maintenance of intestinal homeostasis and protect the intestinal epithelium from injurious stimuli. To address this question, we aimed to isolate the cytoprotective compound from a lactobacillus strain, Lactobacillus brevis SBC8803 which possess the ability to induce cytoprotective heat shock proteins in mouse small intestine. L. brevis was incubated in MRS broth and the supernatant was passed through with a 0.2-µm filter. Caco2/bbe cells were treated with the culture supernatant, and HSP27 expression was evaluated by Western blotting. HSP27-inducible components were separated by ammonium sulfate precipitation, DEAE anion exchange chromatography, gel filtration, and HPLC. Finally, we identified that the HSP27-inducible fraction was polyphosphate (poly P, a simple repeated structure of phosphates, which is a common product of lactobacilli and other bacteria associated with intestinal microflora without any definitive physiological functions. Then, poly P was synthesized by poly P-synthesizing enzyme polyphosphate kinase. The synthesized poly P significantly induced HSP27 from Caco2/BBE cells. In addition, Poly P suppressed the oxidant-induced intestinal permeability in the mouse small intestine and pharmacological inhibitors of p38 MAPK and integrins counteract its protective effect. Daily intrarectal administration of poly P (10 µg improved the inflammation grade and survival rate in 4% sodium dextran sulfate-administered mice. This study, for the first time, demonstrated that poly P is the molecule responsible for maintaining intestinal barrier actions which are mediated through the intestinal integrin β1-p38 MAPK.

  14. Caco-2 accumulation of lutein is greater from human milk than from infant formula despite similar bioaccessibility.

    Science.gov (United States)

    Lipkie, Tristan E; Banavara, Dattatreya; Shah, Bhavini; Morrow, Ardythe L; McMahon, Robert J; Jouni, Zeina E; Ferruzzi, Mario G

    2014-10-01

    Clinical evidence suggests that the bioavailability of lutein is lower from infant formula than from human milk. The purpose of this study was to assess characteristics of human milk and lutein-fortified infant formula that may impact carotenoid delivery. Carotenoid bioaccessibility and intestinal absorption were modeled by in vitro digestion coupled with Caco-2 human intestinal cell culture. Twelve human milk samples were assessed from 1-6 months postpartum, and 10 lutein-fortified infant formula samples from three lutein sources in both ready-to-use and reconstituted powder forms. The relative bioaccessibility of lutein was not different (p > 0.05) between human milk (29 ± 2%) and infant formula (36 ± 4%). However, lutein delivery was 4.5 times greater from human milk than infant formula when including Caco-2 accumulation efficiency. Caco-2 accumulation of lutein was increasingly efficient with decreasing concentration of lutein from milk. Carotenoid bioaccessibility and Caco-2 accumulation were not affected by lactation stage, total lipid content, lutein source, or form of infant formula (powder vs. liquid). These data suggest that the bioavailability of carotenoids is greater from human milk than infant formula primarily due to intestinal absorptive processes, and that absorption of lutein is potentiated by factors from human milk especially at low lutein concentration. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Accelerated Caco-2 cell permeability model for drug discovery.

    Science.gov (United States)

    Sevin, E; Dehouck, L; Fabulas-da Costa, A; Cecchelli, R; Dehouck, M P; Lundquist, S; Culot, M

    2013-01-01

    By culturing Caco-2 cells according to a new and optimized protocol, it has been possible to accelerate the cell culture process in such a way that the cells can be used for experiments after only 6 days. The accelerated Caco-2 model has been compared to the traditional model (requiring 21-25 days of culture) in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, active efflux and to discriminate P-gp substrates. In the new protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with puromycin. The initial cell seeding density was increased two times compared to the traditional procedure and the presence of a low concentration of puromycin in the culture medium reduced the Caco-2 permeability of mannitol. Bi-directional studies were performed with known P-gp substrates (rhodamine 123, digoxin and saquinavir) and with a total of 20 marketed drugs covering a wide range of physicochemical characteristics and therapeutic indications. Strong correlations were obtained between the apparent permeability in absorptive (Papp A→B) or secretory (Papp B→A) of the drugs in the accelerated model and in the traditional models and comparable efflux ratios were observed in the two studied models. The new protocol reduces costs for screening and leads to higher throughput compared to traditional Caco-2 cell models. This accelerated model provides short time-feedback to the drug design during the early stage of drug discovery. © 2013.

  16. Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.

    Science.gov (United States)

    Mills, Dominic C; Gundogdu, Ozan; Elmi, Abdi; Bajaj-Elliott, Mona; Taylor, Peter W; Wren, Brendan W; Dorrell, Nick

    2012-05-01

    Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

  17. Laminin receptor 37/67LR regulates adhesion and proliferation of normal human intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Taoufik Khalfaoui

    Full Text Available Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.

  18. Immunopathology of childhood celiac disease-Key role of intestinal epithelial cells.

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    Pietz, Grzegorz; De, Rituparna; Hedberg, Maria; Sjöberg, Veronika; Sandström, Olof; Hernell, Olle; Hammarström, Sten; Hammarström, Marie-Louise

    2017-01-01

    Celiac disease is a chronic inflammatory disease of the small intestine mucosa due to permanent intolerance to dietary gluten. The aim was to elucidate the role of small intestinal epithelial cells in the immunopathology of celiac disease in particular the influence of celiac disease-associated bacteria. Duodenal biopsies were collected from children with active celiac disease, treated celiac disease, and clinical controls. Intestinal epithelial cells were purified and analyzed for gene expression changes at the mRNA and protein levels. Two in vitro models for human intestinal epithelium, small intestinal enteroids and polarized tight monolayers, were utilized to assess how interferon-γ, interleukin-17A, celiac disease-associated bacteria and gluten influence intestinal epithelial cells. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were significantly upregulated in intestinal epithelial cells at active celiac disease. Of these genes, 70% were upregulated by interferon-γ via the IRF1 pathway. Most interestingly, IRF1 was also upregulated by celiac disease-associated bacteria. The NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin-18, which induces interferon-γ in intraepithelial lymphocytes, was expressed in intestinal epithelial cells. A key factor in the epithelial reaction in celiac disease appears to be over-expression of IRF1 that could be inherent and/or due to presence of undesirable microbes that act directly on IRF1. Dual activation of IRF1 and IRF1-regulated genes, both directly and via the interleukin-18 dependent inflammasome would drastically enhance the inflammatory response and lead to the pathological situation seen in active celiac disease.

  19. Epithelial adhesion molecules and the regulation of intestinal homeostasis during neutrophil transepithelial migration

    Science.gov (United States)

    Sumagin, Ronen; Parkos, Charles A

    2014-01-01

    Epithelial adhesion molecules play essential roles in regulating cellular function and maintaining mucosal tissue homeostasis. Some form epithelial junctional complexes to provide structural support for epithelial monolayers and act as a selectively permeable barrier separating luminal contents from the surrounding tissue. Others serve as docking structures for invading viruses and bacteria, while also regulating the immune response. They can either obstruct or serve as footholds for the immune cells recruited to mucosal surfaces. Currently, it is well appreciated that adhesion molecules collectively serve as environmental cue sensors and trigger signaling events to regulate epithelial function through their association with the cell cytoskeleton and various intracellular adapter proteins. Immune cells, particularly neutrophils (PMN) during transepithelial migration (TEM), can modulate adhesion molecule expression, conformation, and distribution, significantly impacting epithelial function and tissue homeostasis. This review discusses the roles of key intestinal epithelial adhesion molecules in regulating PMN trafficking and outlines the potential consequences on epithelial function. PMID:25838976

  20. Na+-independent phosphate transport in Caco2BBE cells.

    Science.gov (United States)

    Candeal, Eduardo; Caldas, Yupanqui A; Guillén, Natalia; Levi, Moshe; Sorribas, Víctor

    2014-12-15

    Pi transport in epithelia has both Na(+)-dependent and Na(+)-independent components, but so far only Na(+)-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na(+)-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na(+)-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO4 (2-), HCO3 (-), and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved. Copyright © 2014 the American Physiological Society.

  1. Inflammation and the Intestinal Barrier: Leukocyte–Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair

    Science.gov (United States)

    Luissint, Anny-Claude; Parkos, Charles A.; Nusrat, Asma

    2017-01-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte–epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. PMID:27436072

  2. Bioavailability of iron from spinach using an in vitro/human Caco-2 cell bioassay model

    Science.gov (United States)

    Rutzke, Corinne J.; Glahn, Raymond P.; Rutzke, Michael A.; Welch, Ross M.; Langhans, Robert W.; Albright, Louis D.; Combs, Gerald F Jr; Wheeler, Raymond M.

    2004-01-01

    Spinach (Spinacia oleracea) cv Whitney was tested for iron bioavailabilty using an in vitro human intestinal cell culture ferritin bioassay technique previously developed. Spinach was cultured in a growth chamber for 33 days, harvested, and freeze-dried. Total iron in the samples was an average of 71 micrograms/g dry weight. Spinach was digested in vitro (pepsin and 0.1 M HCl followed by pancreatin and 0.1 M NaHCO3) with and without the addition of supplemental ascorbic acid. Caco-2 cell cultures were used to determine iron bioavailability from the spinach mixtures. Production of the iron-binding protein ferritin in the Caco-2 cells showed the supplemental ascorbic acid doubled bioavailability of iron from spinach. The data show fresh spinach is a poor source of iron, and emphasize the importance of evaluation of whole meals rather than single food items. The data support the usefulness of the in vitro/Caco-2 cell ferritin bioassay model for prescreening of space flight diets for bioavailable iron.

  3. Caco-2 cell acquisition of dietary iron(III invokes a nanoparticulate endocytic pathway.

    Directory of Open Access Journals (Sweden)

    Dora I A Pereira

    Full Text Available Dietary non-heme iron contains ferrous [Fe(II] and ferric [Fe(III] iron fractions and the latter should hydrolyze, forming Fe(III oxo-hydroxide particles, on passing from the acidic stomach to less acidic duodenum. Using conditions to mimic the in vivo hydrolytic environment we confirmed the formation of nanodisperse fine ferrihydrite-like particles. Synthetic analogues of these (~ 10 nm hydrodynamic diameter were readily adherent to the cell membrane of differentiated Caco-2 cells and internalization was visualized using transmission electron microscopy. Moreover, Caco-2 exposure to these nanoparticles led to ferritin formation (i.e., iron utilization by the cells, which, unlike for soluble forms of iron, was reduced (p=0.02 by inhibition of clathrin-mediated endocytosis. Simulated lysosomal digestion indicated that the nanoparticles are readily dissolved under mildly acidic conditions with the lysosomal ligand, citrate. This was confirmed in cell culture as monensin inhibited Caco-2 utilization of iron from this source in a dose dependent fashion (p<0.05 whilet soluble iron was again unaffected. Our findings reveal the possibility of an endocytic pathway for acquisition of dietary Fe(III by the small intestinal epithelium, which would complement the established DMT-1 pathway for soluble Fe(II.

  4. pH-dependent and carrier-mediated transport of salicylic acid across Caco-2 cells.

    Science.gov (United States)

    Takanaga, H; Tamai, I; Tsuji, A

    1994-07-01

    The transport of monocarboxylic acid drugs such as salicylic acid was examined in the human colon adenocarcinoma cell line, Caco-2 cells that possess intestinal epithelia-like properties. [14C]Salicylic acid transport was pH-dependent and appeared to follow the pH-partition hypothesis. However, 10 mM unlabelled salicylic acid significantly reduced the permeability coefficient of [14C]salicylic acid. Kinetic analysis of the concentration dependence of the permeation rate of salicylic acid across Caco-2 cells showed both saturable (Kt = 5.28 +/- 0.72 mM Jmax = 36.6 +/- 3.54 nmol min-1 (mg protein)-1) and nonsaturable (kd = 0.37 +/- 0.08 microL min-1 (mg protein)-1) processes. The permeation rate of [14C]salicylic acid was competitively inhibited by both acetic acid and benzoic acid, which were demonstrated in our previous studies to be transported in the carrier-mediated-transport mechanism which is responsible for monocarboxylic acids. Furthermore, certain monocarboxylic acids significantly inhibited [14C]salicylic acid transport, whereas salicylamide and dicarboxylic acids such as succinic acid did not. From these results, it was concluded that the transcellular transport of [14C]salicylic acid across Caco-2 cells is by the pH-dependent and carrier-mediated transport mechanism specific for monocarboxylic acids.

  5. Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

    Science.gov (United States)

    DeSmet, Marsha L; Fleet, James C

    2017-10-01

    High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Selected Phytochemicals and Culinary Plant Extracts Inhibit Fructose Uptake in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Yurim Lee

    2015-09-01

    Full Text Available This study compared the ability of nine culinary plant extracts containing a wide array of phytochemicals to inhibit fructose uptake and then explored the involvement of intestinal fructose transporters and phytochemicals for selected samples. The chemical signature was characterized by high performance liquid chromatography with mass spectrometry. Inhibition of [14C]-fructose uptake was tested by using human intestinal Caco-2 cells. Then, the relative contribution of the two apical-facing intestinal fructose transporters, GLUT2 and GLUT5, and the signature components for fructose uptake inhibition was confirmed in naive, phloretin-treated and forskolin-treated Caco-2 cells. HPLC/MS analysis of the chemical signature revealed that guava leaf contained quercetin and catechin, and turmeric contained curcumin, bisdemethoxycurcumin and dimethoxycurcumin. Similar inhibition of fructose uptake (by ~50% was observed with guava leaf and turmeric in Caco-2 cells, but with a higher contribution of GLUT2 for turmeric and that of GLUT5 for guava leaf. The data suggested that, in turmeric, demethoxycurcumin specifically contributed to GLUT2-mediated fructose uptake inhibition, and curcumin did the same to GLUT5-mediated fructose uptake inhibition, but GLUT2 inhibition was more potent. By contrast, in guava leaf, catechin specifically contributed to GLUT5-mediated fructose uptake inhibition, and quercetin affected both GLUT5- and GLUT2-mediated fructose uptake inhibition, resulting in the higher contribution of GLUT5. These results suggest that demethoxycurcumin is an important contributor to GLUT2-mediated fructose uptake inhibition for turmeric extract, and catechin is the same to GLUT5-mediated fructose uptake inhibition for guava leaf extract. Quercetin, curcumin and bisdemethoxycurcumin contributed to both GLUT5- and GLUT2-mediated fructose uptake inhibition, but the contribution to GLUT5 inhibition was higher than the contribution to GLUT2 inhibition.

  7. Selected Phytochemicals and Culinary Plant Extracts Inhibit Fructose Uptake in Caco-2 Cells.

    Science.gov (United States)

    Lee, Yurim; Lim, Yeni; Kwon, Oran

    2015-09-18

    This study compared the ability of nine culinary plant extracts containing a wide array of phytochemicals to inhibit fructose uptake and then explored the involvement of intestinal fructose transporters and phytochemicals for selected samples. The chemical signature was characterized by high performance liquid chromatography with mass spectrometry. Inhibition of [(14)C]-fructose uptake was tested by using human intestinal Caco-2 cells. Then, the relative contribution of the two apical-facing intestinal fructose transporters, GLUT2 and GLUT5, and the signature components for fructose uptake inhibition was confirmed in naive, phloretin-treated and forskolin-treated Caco-2 cells. HPLC/MS analysis of the chemical signature revealed that guava leaf contained quercetin and catechin, and turmeric contained curcumin, bisdemethoxycurcumin and dimethoxycurcumin. Similar inhibition of fructose uptake (by ~50%) was observed with guava leaf and turmeric in Caco-2 cells, but with a higher contribution of GLUT2 for turmeric and that of GLUT5 for guava leaf. The data suggested that, in turmeric, demethoxycurcumin specifically contributed to GLUT2-mediated fructose uptake inhibition, and curcumin did the same to GLUT5-mediated fructose uptake inhibition, but GLUT2 inhibition was more potent. By contrast, in guava leaf, catechin specifically contributed to GLUT5-mediated fructose uptake inhibition, and quercetin affected both GLUT5- and GLUT2-mediated fructose uptake inhibition, resulting in the higher contribution of GLUT5. These results suggest that demethoxycurcumin is an important contributor to GLUT2-mediated fructose uptake inhibition for turmeric extract, and catechin is the same to GLUT5-mediated fructose uptake inhibition for guava leaf extract. Quercetin, curcumin and bisdemethoxycurcumin contributed to both GLUT5- and GLUT2-mediated fructose uptake inhibition, but the contribution to GLUT5 inhibition was higher than the contribution to GLUT2 inhibition.

  8. Effects of propofol on damage of rat intestinal epithelial cells induced by heat stress and lipopolysaccharides

    Energy Technology Data Exchange (ETDEWEB)

    Tang, J.; Jiang, Y. [Southern Medical University, Nanfang Hospital, Department of Anesthesia, Guangzhou, China, Department of Anesthesia, Nanfang Hospital, Southern Medical University, Guangzhou (China); Tang, Y.; Chen, B. [Guangzhou General Hospital of Guangzhou Military Command, Department of Intensive Care Unit, Guangzhou, China, Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou (China); Sun, X. [Laboratory of Traditional Chinese Medicine Syndrome, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou (China); Su, L.; Liu, Z. [Guangzhou General Hospital of Guangzhou Military Command, Department of Intensive Care Unit, Guangzhou, China, Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou (China)

    2013-06-25

    Gut-derived endotoxin and pathogenic bacteria have been proposed as important causative factors of morbidity and death during heat stroke. However, it is still unclear what kind of damage is induced by heat stress. In this study, the rat intestinal epithelial cell line (IEC-6) was treated with heat stress or a combination of heat stress and lipopolysaccharide (LPS). In addition, propofol, which plays an important role in anti-inflammation and organ protection, was applied to study its effects on cellular viability and apoptosis. Heat stress, LPS, or heat stress combined with LPS stimulation can all cause intestinal epithelial cell damage, including early apoptosis and subsequent necrosis. However, propofol can alleviate injuries caused by heat stress, LPS, or the combination of heat stress and LPS. Interestingly, propofol can only mitigate LPS-induced intestinal epithelial cell apoptosis, and has no protective role in heat-stress-induced apoptosis. This study developed a model that can mimic the intestinal heat stress environment. It demonstrates the effects on intestinal epithelial cell damage, and indicated that propofol could be used as a therapeutic drug for the treatment of heat-stress-induced intestinal injuries.

  9. Intestinal Epithelial-Derived TAK1 Signaling Is Essential for Cytoprotection against Chemical-Induced Colitis

    Science.gov (United States)

    Kim, Jae-Young; Kajino-Sakamoto, Rie; Omori, Emily; Jobin, Christian; Ninomiya-Tsuji, Jun

    2009-01-01

    Background We have previously reported that intestinal epithelium-specific TAK1 deleted mice exhibit severe inflammation and mortality at postnatal day 1 due to TNF-induced epithelial cell death. Although deletion of TNF receptor 1 (TNFR1) can largely rescue those neonatal phenotypes, mice harboring double deletion of TNF receptor 1 (TNFR1) and intestinal epithelium-specific deletion of TAK1 (TNFR1KO/TAK1IEKO) still occasionally show increased inflammation. This indicates that TAK1 is important for TNF-independent regulation of intestinal integrity. Methodology/Principal Findings In this study, we investigated the TNF-independent role of TAK1 in the intestinal epithelium. Because the inflammatory conditions were sporadically developed in the double mutant TNFR1KO/TAK1IEKO mice, we hypothesize that epithelial TAK1 signaling is important for preventing stress-induced barrier dysfunction. To test this hypothesis, the TNFR1KO/TAK1IEKO mice were subjected to acute colitis by administration of dextran sulfate sodium (DSS). We found that loss of TAK1 significantly augments DSS-induced experimental colitis. DSS induced weight loss, intestinal damages and inflammatory markers in TNFR1KO/TAK1IEKO mice at higher levels compared to the TNFR1KO control mice. Apoptosis was strongly induced and epithelial cell proliferation was decreased in the TAK1-deficient intestinal epithelium upon DSS exposure. These suggest that epithelial-derived TAK1 signaling is important for cytoprotection and repair against injury. Finally, we showed that TAK1 is essential for interleukin 1- and bacterial components-induced expression of cytoprotective factors such as interleukin 6 and cycloxygenase 2. Conclusions Homeostatic cytokines and microbes-induced intestinal epithelial TAK1 signaling regulates cytoprotective factors and cell proliferation, which is pivotal for protecting the intestinal epithelium against injury. PMID:19234607

  10. Intestinal epithelial-derived TAK1 signaling is essential for cytoprotection against chemical-induced colitis.

    Directory of Open Access Journals (Sweden)

    Jae-Young Kim

    Full Text Available We have previously reported that intestinal epithelium-specific TAK1 deleted mice exhibit severe inflammation and mortality at postnatal day 1 due to TNF-induced epithelial cell death. Although deletion of TNF receptor 1 (TNFR1 can largely rescue those neonatal phenotypes, mice harboring double deletion of TNF receptor 1 (TNFR1 and intestinal epithelium-specific deletion of TAK1 (TNFR1KO/TAK1(IEKO still occasionally show increased inflammation. This indicates that TAK1 is important for TNF-independent regulation of intestinal integrity.In this study, we investigated the TNF-independent role of TAK1 in the intestinal epithelium. Because the inflammatory conditions were sporadically developed in the double mutant TNFR1KO/TAK1(IEKO mice, we hypothesize that epithelial TAK1 signaling is important for preventing stress-induced barrier dysfunction. To test this hypothesis, the TNFR1KO/TAK1(IEKO mice were subjected to acute colitis by administration of dextran sulfate sodium (DSS. We found that loss of TAK1 significantly augments DSS-induced experimental colitis. DSS induced weight loss, intestinal damages and inflammatory markers in TNFR1KO/TAK1(IEKO mice at higher levels compared to the TNFR1KO control mice. Apoptosis was strongly induced and epithelial cell proliferation was decreased in the TAK1-deficient intestinal epithelium upon DSS exposure. These suggest that epithelial-derived TAK1 signaling is important for cytoprotection and repair against injury. Finally, we showed that TAK1 is essential for interleukin 1- and bacterial components-induced expression of cytoprotective factors such as interleukin 6 and cycloxygenase 2.Homeostatic cytokines and microbes-induced intestinal epithelial TAK1 signaling regulates cytoprotective factors and cell proliferation, which is pivotal for protecting the intestinal epithelium against injury.

  11. Oleic Acid Uptake Reveals the Rescued Enterocyte Phenotype of Colon Cancer Caco-2 by HT29-MTX Cells in Co-Culture Mode.

    Science.gov (United States)

    Berger, Emmanuelle; Nassra, Merian; Atgié, Claude; Plaisancié, Pascale; Géloën, Alain

    2017-07-20

    Gastrointestinal epithelium is the unique route for nutrients and for many pharmaceuticals to enter the body. The present study aimed to analyze precisely whether co-culture of two colon cancer cell lines, mucus-producing cells HT29-MTX and enterocyte-like Caco-2 cells, ameliorate differentiation into an in vitro intestinal barrier model and the signaling pathways involved. Differentiated Caco-2 cells gene datasets were compared first to intestinal or cancer phenotypes and second to signaling pathway gene datasets. Experimental validations were performed in real-time experiments, immunochemistry, and gene expression analyses on Caco-2 versus co-cultures of Caco-2 and HT29-MTX (10%) cells. Partial maintenance of cancer-cell phenotype in differentiated Caco-2 cells was confirmed and fatty acids merged as potential regulators of cancer signaling pathways. HT29-MTX cells induced morphological changes in Caco-2 cells, slightly increased their proliferation rate and profoundly modified gene transcription of phenotype markers, fatty acid receptors, intracellular transporters, and lipid droplet components as well as functional responses to oleic acid. In vitro, enterocyte phenotype was rescued partially by co-culture of cancer cells with goblet cells and completed through oleic acid interaction with signaling pathways dysregulated in cancer cells.

  12. Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells.

    Science.gov (United States)

    Kim, Bohkyung; Bae, Minkyung; Park, Young-Ki; Ma, Hang; Yuan, Tao; Seeram, Navindra P; Lee, Ji-Young

    2017-07-17

    We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. Caco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects. BCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.

  13. The role of ER stress response on ionizing radiation-induced apoptosis in intestinal epithelial cells

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    Lee, Eun Sang; Kim, Kwang Seok; Woo, Sang Keun; Lee, Yong Jin; Jeong, Jae Hoon; Lee, Yoon Jin; Kang, Seong Man; Lim, Young Bin [Laboratory of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2014-04-15

    Apoptosis in the intestinal epithelium is the primary pathologic factor that initiates radiation-induced intestinal injury. However, mechanism involved in ionizing radiation (IR)-induced apoptosis in the intestinal epithelium is not clearly understood. The endoplasmic reticulum (ER) stress is triggered by perturbation of the ER functions, leading to the activation of the unfolded protein response (UPR), an adaptive signaling cascade aimed at restoring ER homeostasis by facilitating the degradation of misfolded proteins and expanding the protein folding capacity of the cell. Recently, IR has also been shown to induce ER stress, thereby activating the UPR signaling pathway in intestinal epithelial cells. In this study, we report the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhance IR-induced caspase3 activation. Knockdown of xbp1 or atf6 with siRNA leads to inhibition of IR-induced caspase3 activation. Taken together, our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Our findings could contribute to the development of new strategies based on modulating ER stress responses to prevent IR-induced intestinal injury.

  14. Modulation of Cytochrome P450 Metabolism and Transport across Intestinal Epithelial Barrier by Ginger Biophenolics

    Science.gov (United States)

    Yang, Chunhua; Donthamsetty, Shashikiran; Cantuaria, Guilherme; Jadhav, Gajanan R.; Vangala, Subrahmanyam; Reid, Michelle D.; Aneja, Ritu

    2014-01-01

    Natural and complementary therapies in conjunction with mainstream cancer care are steadily gaining popularity. Ginger extract (GE) confers significant health-promoting benefits owing to complex additive and/or synergistic interactions between its bioactive constituents. Recently, we showed that preservation of natural “milieu” confers superior anticancer activity on GE over its constituent phytochemicals, 6-gingerol (6G), 8-gingerol (8G), 10-gingerol (10G) and 6-shogaol (6S), through enterohepatic recirculation. Here we further evaluate and compare the effects of GE and its major bioactive constituents on cytochrome P450 (CYP) enzyme activity in human liver microsomes by monitoring metabolites of CYP-specific substrates using LC/MS/MS detection methods. Our data demonstrate that individual gingerols are potent inhibitors of CYP isozymes, whereas GE exhibits a much higher half-maximal inhibition value, indicating no possible herb-drug interactions. However, GE's inhibition of CYP1A2 and CYP2C8 reflects additive interactions among the constituents. In addition, studies performed to evaluate transporter-mediated intestinal efflux using Caco-2 cells revealed that GE and its phenolics are not substrates of P-glycoprotein (Pgp). Intriguingly, however, 10G and 6S were not detected in the receiver compartment, indicating possible biotransformation across the Caco-2 monolayer. These data strengthen the notion that an interplay of complex interactions among ginger phytochemicals when fed as whole extract dictates its bioactivity highlighting the importance of consuming whole foods over single agents. Our study substantiates the need for an in-depth analysis of hepatic biotransformation events and distribution profiles of GE and its active phenolics for the design of safe regimens. PMID:25251219

  15. Modulation of cytochrome P450 metabolism and transport across intestinal epithelial barrier by ginger biophenolics.

    Directory of Open Access Journals (Sweden)

    Rao Mukkavilli

    Full Text Available Natural and complementary therapies in conjunction with mainstream cancer care are steadily gaining popularity. Ginger extract (GE confers significant health-promoting benefits owing to complex additive and/or synergistic interactions between its bioactive constituents. Recently, we showed that preservation of natural "milieu" confers superior anticancer activity on GE over its constituent phytochemicals, 6-gingerol (6G, 8-gingerol (8 G, 10-gingerol (10 G and 6-shogaol (6S, through enterohepatic recirculation. Here we further evaluate and compare the effects of GE and its major bioactive constituents on cytochrome P450 (CYP enzyme activity in human liver microsomes by monitoring metabolites of CYP-specific substrates using LC/MS/MS detection methods. Our data demonstrate that individual gingerols are potent inhibitors of CYP isozymes, whereas GE exhibits a much higher half-maximal inhibition value, indicating no possible herb-drug interactions. However, GE's inhibition of CYP1A2 and CYP2C8 reflects additive interactions among the constituents. In addition, studies performed to evaluate transporter-mediated intestinal efflux using Caco-2 cells revealed that GE and its phenolics are not substrates of P-glycoprotein (Pgp. Intriguingly, however, 10 G and 6S were not detected in the receiver compartment, indicating possible biotransformation across the Caco-2 monolayer. These data strengthen the notion that an interplay of complex interactions among ginger phytochemicals when fed as whole extract dictates its bioactivity highlighting the importance of consuming whole foods over single agents. Our study substantiates the need for an in-depth analysis of hepatic biotransformation events and distribution profiles of GE and its active phenolics for the design of safe regimens.

  16. Identification of Epithelial to Mesenchymal Transition as a Novel Source of Fibroblasts in Intestinal Fibrosis*

    Science.gov (United States)

    Flier, Sarah N.; Tanjore, Harikrishna; Kokkotou, Efi G.; Sugimoto, Hikaru; Zeisberg, Michael; Kalluri, Raghu

    2010-01-01

    Intestinal fibrosis is a major complication of Crohn disease (CD), but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD and also human inflammatory bowel disease. Mice administered intrarectal 2,4,6-trinitrobenzene sulfonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin+ intestinal epithelial cells activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells that express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, the appearance of LacZ+ cells that also express the fibroblast marker FSP1 unequivocally demonstrates EMT. Transforming growth factor (TGF)-β1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells in vitro, and bone morphogenic protein-7, an antagonist of TGF-β1, inhibits EMT and fibrosis both in vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn colitis and that inhibition of TGF-β1 with recombinant human bone morphogenic protein-7 prevents this process and prevents fibrosis. PMID:20363741

  17. Identification of epithelial to mesenchymal transition as a novel source of fibroblasts in intestinal fibrosis.

    Science.gov (United States)

    Flier, Sarah N; Tanjore, Harikrishna; Kokkotou, Efi G; Sugimoto, Hikaru; Zeisberg, Michael; Kalluri, Raghu

    2010-06-25

    Intestinal fibrosis is a major complication of Crohn disease (CD), but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD and also human inflammatory bowel disease. Mice administered intrarectal 2,4,6-trinitrobenzene sulfonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin(+) intestinal epithelial cells activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells that express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, the appearance of LacZ(+) cells that also express the fibroblast marker FSP1 unequivocally demonstrates EMT. Transforming growth factor (TGF)-beta1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells in vitro, and bone morphogenic protein-7, an antagonist of TGF-beta1, inhibits EMT and fibrosis both in vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn colitis and that inhibition of TGF-beta1 with recombinant human bone morphogenic protein-7 prevents this process and prevents fibrosis.

  18. O-antigen delays lipopolysaccharide recognition and impairs antibacterial host defense in murine intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Claudia U Duerr

    2009-09-01

    Full Text Available Although Toll-like receptor (TLR 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs. Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S. Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.

  19. Intestinal Trefoil Factor/TFF3 Promotes Re-epithelialization of Corneal Wounds*

    OpenAIRE

    Paulsen, Friedrich P.; Woon, Chee-Wai; Varoga, Deike; Jansen, Anne; Garreis, Fabian; Jäger, Kristin; Amm, Marita; Podolsky, Daniel K.; Steven, Philipp; Barker, Nicholas P.; Sel, Saadettin

    2008-01-01

    Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed ...

  20. Advanced three-dimensional culture of equine intestinal epithelial stem cells.

    Science.gov (United States)

    Stewart, A Stieler; Freund, J M; Gonzalez, L M

    2017-08-09

    Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. Descriptive study. Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed. Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease

  1. Identification of Epithelial to Mesenchymal Transition as a Novel Source of Fibroblasts in Intestinal Fibrosis*

    OpenAIRE

    Flier, Sarah N.; Tanjore, Harikrishna; Kokkotou, Efi G.; Sugimoto, Hikaru; Zeisberg, Michael; Kalluri, Raghu

    2010-01-01

    Intestinal fibrosis is a major complication of Crohn disease (CD), but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD and also human inflammatory bowel disease. Mice administered intrarectal 2,4,6-trinitrobenzene sulfonic acid (TNBS) develop inf...

  2. STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing.

    Science.gov (United States)

    Pickert, Geethanjali; Neufert, Clemens; Leppkes, Moritz; Zheng, Yan; Wittkopf, Nadine; Warntjen, Moritz; Lehr, Hans-Anton; Hirth, Sebastian; Weigmann, Benno; Wirtz, Stefan; Ouyang, Wenjun; Neurath, Markus F; Becker, Christoph

    2009-07-06

    Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c(+) cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3(IEC-KO) mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing.

  3. Functionalized carbon nanomaterials: exploring the interactions with Caco-2 cells for potential oral drug delivery

    Science.gov (United States)

    Coyuco, Jurja C; Liu, Yuanjie; Tan, Bee-Jen; Chiu, Gigi NC

    2011-01-01

    Although carbon nanomaterials (CNMs) have been increasingly studied for their biomedical applications, there is limited research on these novel materials for oral drug delivery. As such, this study aimed to explore the potential of CNMs in oral drug delivery, and the objectives were to evaluate CNM cytotoxicity and their abilities to modulate paracellular transport and the P-glycoprotein (P-gp) efflux pump. Three types of functionalized CNMs were studied, including polyhydroxy small-gap fullerenes (OH-fullerenes), carboxylic acid functionalized single-walled carbon nanotubes (f SWCNT-COOH) and poly(ethylene glycol) functionalized single-walled carbon nanotubes (f SWCNT-PEG), using the well-established Caco-2 cell monolayer to represent the intestinal epithelium. All three CNMs had minimum cytotoxicity on Caco-2 cells, as demonstrated through lactose dehydrogenase release and 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Of the three CNMs, f SWCNT-COOH significantly reduced transepithelial electrical resistance and enhanced transport of Lucifer Yellow across the Caco-2 monolayer. Confocal fluorescence microscopy showed that f SWCNT-COOH treated cells had the highest perturbation in the distribution of ZO-1, a protein marker of tight junction, suggesting that f SWCNT-COOH could enhance paracellular permeability via disruption of tight junctions. This modulating effect of f SWCNT-COOH can be reversed over time. Furthermore, cellular accumulation of the P-gp substrate, rhodamine-123, was significantly increased in cells treated with f SWCNT-COOH, suggestive of P-gp inhibition. Of note, f SWCNT-PEG could increase rhodamine-123 accumulation without modifying the tight junction. Collectively, these results suggest that the functionalized CNMs could be useful as modulators for oral drug delivery, and the differential effects on the intestinal epithelium imparted by different types of CNMs would create unique opportunities for drug-specific oral

  4. Functionalized carbon nanomaterials: exploring the interactions with Caco-2 cells for potential oral drug delivery.

    Science.gov (United States)

    Coyuco, Jurja C; Liu, Yuanjie; Tan, Bee-Jen; Chiu, Gigi N C

    2011-01-01

    Although carbon nanomaterials (CNMs) have been increasingly studied for their biomedical applications, there is limited research on these novel materials for oral drug delivery. As such, this study aimed to explore the potential of CNMs in oral drug delivery, and the objectives were to evaluate CNM cytotoxicity and their abilities to modulate paracellular transport and the P-glycoprotein (P-gp) efflux pump. Three types of functionalized CNMs were studied, including polyhydroxy small-gap fullerenes (OH-fullerenes), carboxylic acid functionalized single-walled carbon nanotubes (f SWCNT-COOH) and poly(ethylene glycol) functionalized single-walled carbon nanotubes (f SWCNT-PEG), using the well-established Caco-2 cell monolayer to represent the intestinal epithelium. All three CNMs had minimum cytotoxicity on Caco-2 cells, as demonstrated through lactose dehydrogenase release and 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Of the three CNMs, f SWCNT-COOH significantly reduced transepithelial electrical resistance and enhanced transport of Lucifer Yellow across the Caco-2 monolayer. Confocal fluorescence microscopy showed that f SWCNT-COOH treated cells had the highest perturbation in the distribution of ZO-1, a protein marker of tight junction, suggesting that f SWCNT-COOH could enhance paracellular permeability via disruption of tight junctions. This modulating effect of f SWCNT-COOH can be reversed over time. Furthermore, cellular accumulation of the P-gp substrate, rhodamine-123, was significantly increased in cells treated with f SWCNT-COOH, suggestive of P-gp inhibition. Of note, f SWCNT-PEG could increase rhodamine-123 accumulation without modifying the tight junction. Collectively, these results suggest that the functionalized CNMs could be useful as modulators for oral drug delivery, and the differential effects on the intestinal epithelium imparted by different types of CNMs would create unique opportunities for drug-specific oral

  5. TTC7A mutations disrupt intestinal epithelial apicobasal polarity

    NARCIS (Netherlands)

    Bigorgne, Amélie E; Farin, Henner F; Lemoine, Roxane; Mahlaoui, Nizar; Lambert, Nathalie; Gil, Marine; Schulz, Ansgar; Philippet, Pierre; Schlesser, Patrick; Abrahamsen, Tore G; Oymar, Knut; Davies, E Graham; Ellingsen, Christian Lycke; Leteurtre, Emmanuelle; Moreau-Massart, Brigitte; Berrebi, Dominique; Bole-Feysot, Christine; Nischke, Patrick; Brousse, Nicole; Fischer, Alain; Clevers, Hans; de Saint Basile, Geneviève

    Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and

  6. Alisertib Induces Cell Cycle Arrest, Apoptosis, Autophagy and Suppresses EMT in HT29 and Caco-2 Cells

    Science.gov (United States)

    Ren, Bao-Jun; Zhou, Zhi-Wei; Zhu, Da-Jian; Ju, Yong-Le; Wu, Jin-Hao; Ouyang, Man-Zhao; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Colorectal cancer (CRC) is one of the most common malignancies worldwide with substantial mortality and morbidity. Alisertib (ALS) is a selective Aurora kinase A (AURKA) inhibitor with unclear effect and molecular interactome on CRC. This study aimed to evaluate the molecular interactome and anticancer effect of ALS and explore the underlying mechanisms in HT29 and Caco-2 cells. ALS markedly arrested cells in G2/M phase in both cell lines, accompanied by remarkable alterations in the expression level of key cell cycle regulators. ALS induced apoptosis in HT29 and Caco-2 cells through mitochondrial and death receptor pathways. ALS also induced autophagy in HT29 and Caco-2 cells, with the suppression of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), but activation of 5′ AMP-activated protein kinase (AMPK) signaling pathways. There was a differential modulating effect of ALS on p38 MAPK signaling pathway in both cell lines. Moreover, induction or inhibition of autophagy modulated basal and ALS-induced apoptosis in both cell lines. ALS potently suppressed epithelial to mesenchymal transition (EMT) in HT29 and Caco-2 cells. Collectively, it suggests that induction of cell cycle arrest, promotion of apoptosis and autophagy, and suppression of EMT involving mitochondrial, death receptor, PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways contribute to the cancer cell killing effect of ALS on CRC cells. PMID:26729093

  7. Gut microbial colonization orchestrates TLR2 expression, signaling and epithelial proliferation in the small intestinal mucosa.

    Directory of Open Access Journals (Sweden)

    Nives Hörmann

    Full Text Available The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs. Here, we report that colonization of germ-free (GF Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2 and protein-kinase B (AKT induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.

  8. The Potential Health Benefits of Polyphenol-Rich Extracts from Cichorium intybus L. Studied on Caco-2 Cells Model

    Directory of Open Access Journals (Sweden)

    Elena Azzini

    2016-01-01

    Full Text Available Phytochemicals can exert their bioactivity without reaching the systemic circulation; scarcely absorbed antioxidants might reach the large bowel contributing to protection from oxidative damage-induced gastrointestinal diseases. In the present work, we aimed to study the relationship between potential activity of polyphenol-rich extracts from Cichorium intybus L. and changes in morphological characteristics on Caco-2 cells. Phytochemicals content (carotenoids and flavonoids and total antioxidant activity of Red Chicory of Treviso and Variegated Chicory of Castelfranco were evaluated. The bioactivity of polyphenol-rich extracts from chicories was studied in in vitro Caco-2 cell monolayers model. Morphological characteristics changes to test the antioxidant and/or prooxidant effect were verified by histological analysis and observed by Electronic Scansion Microscopy (SEM. On Caco-2 cell model, the polyphenols fractions from chicories have indicated a moderate antioxidant behavior until 17 μM concentration, while 70 μM and 34 μM exert cytotoxic effects for Treviso’s and Castelfranco’s Chicory, respectively, highlighted by TEER decreasing, increased permeability, and alteration of epithelium. Our findings support the beneficial effects of these products in counteracting the oxidative stress and cellular damage, induced in vitro on Caco-2 cell model, through interaction with the mucopolysaccharide complexes in the glycocalyx, maintaining in vivo a healthy and effective intestinal barrier.

  9. The Potential Health Benefits of Polyphenol-Rich Extracts from Cichorium intybus L. Studied on Caco-2 Cells Model

    Science.gov (United States)

    Azzini, Elena; Maiani, Giuseppe; Garaguso, Ivana; Polito, Angela; Foddai, Maria S.; Venneria, Eugenia; Durazzo, Alessandra; Intorre, Federica; Palomba, Lara; Rauseo, Maria L.; Lombardi-Boccia, Ginevra; Nobili, Fabio

    2016-01-01

    Phytochemicals can exert their bioactivity without reaching the systemic circulation; scarcely absorbed antioxidants might reach the large bowel contributing to protection from oxidative damage-induced gastrointestinal diseases. In the present work, we aimed to study the relationship between potential activity of polyphenol-rich extracts from Cichorium intybus L. and changes in morphological characteristics on Caco-2 cells. Phytochemicals content (carotenoids and flavonoids) and total antioxidant activity of Red Chicory of Treviso and Variegated Chicory of Castelfranco were evaluated. The bioactivity of polyphenol-rich extracts from chicories was studied in in vitro Caco-2 cell monolayers model. Morphological characteristics changes to test the antioxidant and/or prooxidant effect were verified by histological analysis and observed by Electronic Scansion Microscopy (SEM). On Caco-2 cell model, the polyphenols fractions from chicories have indicated a moderate antioxidant behavior until 17 μM concentration, while 70 μM and 34 μM exert cytotoxic effects for Treviso's and Castelfranco's Chicory, respectively, highlighted by TEER decreasing, increased permeability, and alteration of epithelium. Our findings support the beneficial effects of these products in counteracting the oxidative stress and cellular damage, induced in vitro on Caco-2 cell model, through interaction with the mucopolysaccharide complexes in the glycocalyx, maintaining in vivo a healthy and effective intestinal barrier. PMID:26843906

  10. Accumulation and Transport of Roxarsone, Arsenobetaine, and Inorganic Arsenic Using the Human Immortalized Caco-2 Cell Line.

    Science.gov (United States)

    Liu, Qingqing; Leslie, Elaine M; Le, X Chris

    2016-11-23

    Roxarsone (Rox), an organoarsenic compound, served as a feed additive in the poultry industry for more than 60 years. Residual amounts of Rox present in chicken meat could give rise to potential human exposure to Rox. However, studies on the bioavailability of Rox in humans are scarce. We report here the accumulation and transepithelial transport of Rox using the human colon-derived adenocarcinoma cell line (Caco-2) model. The cellular accumulation and transepithelial passage of Rox in Caco-2 cells were evaluated and compared to those of arsenobetaine (AsB), arsenite (AsIII), and arsenate (AsV). When Caco-2 cells were exposed to 3 μM Rox, AsB, and AsIII separately for 24 h, the maximum accumulation was reached at 12 h. After 24-h exposure, the accumulated Rox was 6-20 times less than AsB and AsIII. The permeability of Rox from the apical to basolateral side of Caco-2 monolayers was similar to AsV but less than AsIII and AsB. The results of lower bioavailability of Rox are consistent with previous observations of relatively lower amounts of Rox retained in the breast meat of Rox-fed chickens. These data provide useful information for assessing human exposure to and intestinal bioavailability of Roxarsone.

  11. A pSMAD/CDX2 Complex Is Essential for the Intestinalization of Epithelial Metaplasia

    Directory of Open Access Journals (Sweden)

    Luigi Mari

    2014-05-01

    Full Text Available The molecular mechanisms leading to epithelial metaplasias are poorly understood. Barrett's esophagus is a premalignant metaplastic change of the esophageal epithelium into columnar epithelium, occurring in patients suffering from gastroesophageal reflux disease. Mechanisms behind the development of the intestinal subtype, which is associated with the highest cancer risk, are unclear. In humans, it has been suggested that a nonspecialized columnar metaplasia precedes the development of intestinal metaplasia. Here, we propose that a complex made up of at least two factors needs to be activated simultaneously to drive the expression of intestinal type of genes. Using unique animal models and robust in vitro assays, we show that the nonspecialized columnar metaplasia is a precursor of intestinal metaplasia and that pSMAD/CDX2 interaction is essential for the switch toward an intestinal phenotype.

  12. Protecting intestinal epithelial integrity by galacto-oligosaccharides: Keeping it tight

    NARCIS (Netherlands)

    Akbari, P.

    2016-01-01

    The intestinal barrier serves as a first line of host defense against potentially harmful stressors from the environment ingested with food, and is primarily formed by epithelial cells connected by tight junctions. Oligosaccharides have been identified as components in milk, particularly in

  13. Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels

    NARCIS (Netherlands)

    Sachs, Norman; Tsukamoto, Yoshiyuki; Kujala, Pekka; Peters, Peter J; Clevers, Hans

    2017-01-01

    Multiple recent examples highlight how stem cells can self-organize in vitro to establish organoids that closely resemble their in vivo counterparts. Single Lgr5+ mouse intestinal stem cells can be cultured under defined conditions forming ever-expanding epithelial organoids that retain cell

  14. Camplyobacter jejuni translocation across intestinal epithelial cells is facilitated by ganglioside-like lipooligosaccharide structures.

    NARCIS (Netherlands)

    Nieuwenhuis, E.E.S.; Louwen, R.; Marrewijk, L. van; Horst, D.; Ruiter, L. de; Heikema, A.P.; Wamel, W.J. van; Wagenaar, J.A.; Endtz, H.P.; Samsom, J.N.; Baarlen, P. van; Akhmanova, A.S.; Belkum, A. van

    2012-01-01

    Translocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study we investigated whether sialylation of C.

  15. Campylobacter jejuni Translocation across Intestinal Epithelial Cells Is Facilitated by Ganglioside-Like Lipooligosaccharide Structures

    NARCIS (Netherlands)

    Louwen, R.; Nieuwenhuis, E.E.S.; Marrewijk, van L.; Horst-Kreft, D.; Ruiter, de L.; Heikema, A.P.; Wamel, van W.J.; Wagenaar, J.A.; Endtz, H.P.; Samsom, J.; Baarlen, van P.; Akhmanova, A.; Belkum, van A.

    2012-01-01

    Translocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study, we investigated whether sialylation of C.

  16. Campylobacter jejuni translocation across intestinal epithelial cells is facilitated by ganglioside-like lipooligosaccharide structures

    NARCIS (Netherlands)

    R.P.L. Louwen (Rogier); E.E.S. Nieuwenhuis (Edward); L. van Marrewijk (Leonie); D. Horst-Kreft (Deborah); L.F. de Ruiter (Lilian); A.P. Heikema (Astrid); W.J.B. van Wamel (Willem); J.A. Wagenaar (Jaap); H.P. Endtz (Hubert); J.N. Samsom (Janneke); P. van Baarlen (Peter); A.S. Akhmanova (Anna); A.F. van Belkum (Alex)

    2012-01-01

    textabstractTranslocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study, we investigated whether

  17. Processing of whey modulates proliferative and immune functions in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Nguyen, Duc Ninh; Sangild, Per Torp; Li, Yanqi

    2016-01-01

    of bioactive proteins and effects on proliferation and immune response in intestinal epithelial cells (IEC). The results showed that low-heat-treated WPC had elevated levels of lactoferrin and transforming growth factor-β2 compared with that of standard WPC. The level of aggregates depended on the source...

  18. Mycotoxins modify the barrier function of Caco-2 cells through differential gene expression of specific claudin isoforms: Protective effect of illite mineral clay.

    Science.gov (United States)

    Romero, Alejandro; Ares, Irma; Ramos, Eva; Castellano, Víctor; Martínez, Marta; Martínez-Larrañaga, María-Rosa; Anadón, Arturo; Martínez, María-Aránzazu

    2016-04-15

    Aflatoxin B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA) and T-2 toxin (T2) are mycotoxins that commonly contaminate the food chain and cause various toxicological effects. Their global occurrence is regarded as an important risk factor for human and animal health. In this study, the results demonstrate that, in human Caco-2 cells, AFB1, FB1, OTA and T2 origin cytotoxic effects, determining cell viability through MTT assay and LDH leakage, and decrease trans-epithelial electrical resistance (TEER). The decrease in barrier properties is concomitant with a reduction in the expression levels of the tight junction constituents claudin-3, claudin-4 and occludin. The protective effect of mineral clays (diosmectite, montmorillonite and illite) on alterations in cell viability and epithelial barrier function induced by the mycotoxins was also evaluated. Illite was the best clay to prevent the mycotoxin effects. Illite plus mycotoxin co-treatment completely abolished AFB1 and FB1-induced cytotoxicity. Also, the decreases in the gene expression of claudins and the reduction of TEER induced by mycotoxins were reversed by the illite plus mycotoxin co-treatment. In conclusion, these results demonstrated that mycotoxins AFB1, FB1, T2 and OTA disrupt the intestinal barrier permeability by a mechanism involving reduction of claudin isoform expressions, and illite counteracts this disruption. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. A20 restricts wnt signaling in intestinal epithelial cells and suppresses colon carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Ling Shao

    Full Text Available Colon carcinogenesis consists of a multistep process during which a series of genetic and epigenetic adaptations occur that lead to malignant transformation. Here, we have studied the role of A20 (also known as TNFAIP3, a ubiquitin-editing enzyme that restricts NFκB and cell death signaling, in intestinal homeostasis and tumorigenesis. We have found that A20 expression is consistently reduced in human colonic adenomas than in normal colonic tissues. To further investigate A20's potential roles in regulating colon carcinogenesis, we have generated mice lacking A20 specifically in intestinal epithelial cells and interbred these with mice harboring a mutation in the adenomatous polyposis coli gene (APC(min. While A20(FL/FL villin-Cre mice exhibit uninflamed intestines without polyps, A20(FL/FL villin-Cre APC(min/+ mice contain far greater numbers and larger colonic polyps than control APC(min mice. We find that A20 binds to the β-catenin destruction complex and restricts canonical wnt signaling by supporting ubiquitination and degradation of β-catenin in intestinal epithelial cells. Moreover, acute deletion of A20 from intestinal epithelial cells in vivo leads to enhanced expression of the β-catenin dependent genes cyclinD1 and c-myc, known promoters of colon cancer. Taken together, these findings demonstrate new roles for A20 in restricting β-catenin signaling and preventing colon tumorigenesis.

  20. Defining suitable reference genes for RT-qPCR analysis on intestinal epithelial cells.

    Science.gov (United States)

    Sirakov, Maria; Borra, Marco; Cambuli, Francesca Maria; Plateroti, Michelina

    2013-07-01

    The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs-geNorm and Normfinder-were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.

  1. Digestive Stability and Transport of Norbixin, a 24-Carbon Carotenoid, across Monolayers of Caco-2 Cells

    Science.gov (United States)

    Polar-Cabrera, Karina; Huo, Tianyao; Schwartz, Steven J.; Failla, Mark L.

    2013-01-01

    Annatto is a natural pigment widely used in the food industry to add yellow to red colors to dairy and cereal products. Here, the in vitro bioaccessibility and potential bioavailability of norbixin, the abundant 24-carbon carotenoid in annatto, were investigated. Norbixin added to milk was highly stable during simulated digestion, and bile salts enhanced partitioning of this carotenoid in the aqueous fraction during the small intestinal phase of digestion. Apical uptake of norbixin by Caco-2 cells was proportional to the concentration in apical medium, but cellular content increased only slightly after 60 min. Transport of norbixin to the basolateral compartment was maximum at 120 min. Both all-trans and cis isomers of norbixin were present in cells and basolateral medium. The results suggest that ingested norbixin is stable during gastric and small intestinal phases of digestion and that both cis and all-trans isomers are bioavailable. PMID:20408560

  2. Loss of Kindlin-1 causes skin atrophy and lethal neonatal intestinal epithelial dysfunction.

    Directory of Open Access Journals (Sweden)

    Siegfried Ussar

    2008-12-01

    Full Text Available Kindler Syndrome (KS, characterized by transient skin blistering followed by abnormal pigmentation, skin atrophy, and skin cancer, is caused by mutations in the FERMT1 gene. Although a few KS patients have been reported to also develop ulcerative colitis (UC, a causal link to the FERMT1 gene mutation is unknown. The FERMT1 gene product belongs to a family of focal adhesion proteins (Kindlin-1, -2, -3 that bind several beta integrin cytoplasmic domains. Here, we show that deleting Kindlin-1 in mice gives rise to skin atrophy and an intestinal epithelial dysfunction with similarities to human UC. This intestinal dysfunction results in perinatal lethality and is triggered by defective intestinal epithelial cell integrin activation, leading to detachment of this barrier followed by a destructive inflammatory response.

  3. Enterohemorrhagic Escherichia coli infection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells.

    Science.gov (United States)

    Lukyanenko, Valeriy; Malyukova, Irina; Hubbard, Ann; Delannoy, Michael; Boedeker, Edgar; Zhu, Chengru; Cebotaru, Liudmila; Kovbasnjuk, Olga

    2011-11-01

    Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in ∼10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.

  4. Artemisinin permeability via Caco-2 cells increases after simulated digestion of Artemisia annua leaves.

    Science.gov (United States)

    Desrosiers, Matthew R; Weathers, Pamela J

    2018-01-10

    Artemisia annua has been used for > 2000yrs to treat fever and is more recently known for producing the important antimalarial drug, artemisinin. Artemisinin combination therapies (ACTs) are effective for treating malaria, but are often unavailable to those in need. Dried leaves of A. annua (DLA) have recently been studied as a cost effective alternative to traditional ACTs. DLA was shown to dramatically increase oral bioavailability compared to pure artemisinin, so more investigation into the mechanisms causing this increased bioavailability is needed. In this study, we used a simulated digestion system coupled with Caco-2 cell permeability assays to investigate the intestinal permeability of DLA compared to pure artemisinin. We also determined the effects of different phytochemicals (7 flavonoids, 3 monoterpenes, 2 phenolic acids, scopoletin and inulin) and the cytochrome P450 isoform CYP3A4 on artemisinin intestinal permeability. Artemisinin permeability, when delivered as digested DLA, significantly increased by 37% (Papp = 8.03 × 10-5cms-1) compared to pure artemisinin (Papp = 5.03 × 10-5cms-1). However, none of the phytochemicals tested or CYP3A4 had any significant effect on the intestinal permeability of artemisinin. We also showed that essential oil derived from A. annua negatively affected the intestinal permeability of artemisinin, but only after simulated digestion. Finally, we showed that A. annua essential oil reduced the transepithelial electrical resistance of Caco-2 monolayers, but only in the presence of bile. Although also reduced by essential oils, artemisinin Papp subsequently recovered in the presence of plant matrix. These results shed light on the mechanisms by which DLA enhances the oral bioavailability of artemisinin. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Plasticity of intestinal epithelial cells in regeneration and cancer

    NARCIS (Netherlands)

    Tetteh, Paul W.

    2015-01-01

    Cellular plasticity refers to the ability of a cell to change its fate or identity in response to external or intrinsic factors. Regeneration of the intestinal epithelium after injury is driven mainly by plasticity of crypt stem cells that can rapidly divide to replace all the lost cells. Stem cell

  6. Permeability and modulation of the intestinal epithelial barrier in vitro

    NARCIS (Netherlands)

    Duizer, E.

    1999-01-01

    The bioavailability of all ingested compounds is to a great extend determined by the ability of these compounds to pass the intestinal epithelium. A high bioavailability is guaranteed for most nutrients and electrolytes since they are actively absorbed by the epithelium. The same

  7. Epithelial structure and function in the hen lower intestine

    DEFF Research Database (Denmark)

    Laverty, G.; Elbrønd, Vibeke Sødring; Árnason, Sigvatur S.

    2006-01-01

    In birds, transport processes in the lower intestine mediate absorption of ions, water and a variety of organic substrates, including significant amounts of glucose, amino acids derived from protein associated with urate spheres, and short-chain fatty acids derived from fermentation processes. Th...

  8. Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages.

    Science.gov (United States)

    Yasuda, Michiko; Furuyashiki, Takashi; Nakamura, Toshiyuki; Kakutani, Ryo; Takata, Hiroki; Ashida, Hitoshi

    2013-09-01

    Previously, we developed enzymatically synthesized glycogen (ESG) from starch, and showed its immunomodulatory and dietary fiber-like activities. In this study, we investigated the metabolism of ESG and its immunomodulatory activity using differentiated Caco-2 cells as a model of the intestinal barrier. In a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages, mRNA expression of IL-6, IL-8, IL-1β and BAFF cytokines was up-regulated in Caco-2 cells and IL-8 production in basolateral medium was induced after 24 h apical treatment with 5 mg ml(-1) of ESG. The mRNA level of iNOS was also up-regulated in RAW264.7 macrophages. After characterization of the binding of anti-glycogen monoclonal antibodies (IV58B6 and ESG1A9) to ESG and its digested metabolite resistant glycogen (RG), an enzyme-linked immunosorbent assay (ELISA) system was developed to quantify ESG and RG. Using this system, we investigated the metabolism of ESG in differentiated Caco-2 cells. When ESG (7000 kDa, 5 mg ml(-1)) was added to the apical side of Caco-2 monolayers, ESG disappeared and RG (about 3000 kDa, 3.5 mg ml(-1)) appeared in the apical solution during a 24 h incubation. Neither ESG nor RG was detected in the basolateral solution. In addition, both ESG and RG were bound to TLR2 in Caco-2 cells. In conclusion, we suggest that ESG is metabolized to a RG-like structure in the intestine, and this metabolite activates the immune system via stimulation of the intestinal epithelium, although neither ESG nor its metabolite could permeate the intestinal cells under our experimental conditions. These results provide evidence for the beneficial function of ESG as a food ingredient.

  9. Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state.

    Science.gov (United States)

    Kämpfer, Angela A M; Urbán, Patricia; Gioria, Sabrina; Kanase, Nilesh; Stone, Vicki; Kinsner-Ovaskainen, Agnieszka

    2017-12-01

    The intestine forms the largest interface between the environment and the human organism. Its integrity and functioning are crucial for the uptake of nutrients while preventing access of harmful antigens. Inflammatory conditions can significantly change the normal functioning of the intestine. In vitro models that adequately reproduce both healthy and inflamed intestinal tissue could provide a useful tool for studying the mechanisms of intestinal inflammation and investigating new therapeutic drugs. We established a co-culture of Caco-2 and PMA-differentiated THP-1 cells that mimics the intestine in healthy and controlled inflamed states. In homoeostatic conditions without stimulation, Caco-2 and THP-1 cells were co-cultured for 48h without affecting the barrier integrity and with no increase in the release of cytokines, nitric oxide or lactate dehydrogenase. To simulate the inflamed intestine, the Caco-2 barrier was primed with IFN-γ and THP-1 cells were pre-stimulated with LPS and IFN-γ. In these conditions a significant but temporary reduction in barrier integrity was measured, and large concentrations of pro-inflammatory cytokines and cytotoxicity markers detected. With its ability to feature numerous hallmarks of intestinal inflammation the presented co-culture model of epithelial cells and macrophages offers a unique possibility to study exposure effects in relation to the health status of the intestine. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Silibinin attenuates radiation-induced intestinal fibrosis and reverses epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Kim, Joong Sun; Han, Na-Kyung; Kim, Sung-Ho; Lee, Hae-June

    2017-09-19

    Radiotherapy is a common treatment for cancer patients, but its use is often restricted by the tolerance of normal tissue. As cancer patients live longer, delayed radiation effects on normal tissue have become a concern. Radiation-induced enteropathy, including inflammatory bowel disease and fibrosis, are major issues for long-term cancer survivors. To investigate whether silibinin attenuates delayed radiation-induced intestinal injury in mice, we focused on intestinal fibrotic changes. Silibinin improved delayed radiation injuries in mice in association with decreased collagen deposition within the intestines and deceased transforming growth factor (TGF)-β1 levels in the intestine and plasma. Treating mice bearing CT26 mouse colon cancer tumors with both silibinin and radiation stimulated tumor regression more than radiation alone. We also investigated the effect of silibinin on the radiation-induced epithelial-to-mesenchymal transition (EMT), the primary mechanism of fibrosis. We assessed changes in E-cadherin, N-cadherin, and α-smooth muscle actin expression, and demonstrated that silibinin attenuates radiation-induced EMT. Irradiating intestinal epithelial cells increased TGF-β1 levels, but silibinin suppressed TGF-β1 expression by inhibiting Smad2/3 phosphorylation. These results suggest silibinin has the potential to serve as a useful therapeutic agent in patients with radiation-induced intestinal fibrosis.

  11. Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG

    Directory of Open Access Journals (Sweden)

    Burkholder Kristin M

    2009-07-01

    Full Text Available Abstract Background Physiological stressors may alter susceptibility of the host intestinal epithelium to infection by enteric pathogens. In the current study, cytotoxic effect, adhesion and invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium to Caco-2 cells exposed to thermal stress (41°C, 1 h was investigated. Probiotic bacteria have been shown to reduce interaction of pathogens with the epithelium under non-stress conditions and may have a significant effect on epithelial viability during infection; however, probiotic effect on pathogen interaction with epithelial cells under physiological stress is not known. Therefore, we investigated the influence of Lactobacillus rhamnosus GG and Lactobacillus gasseri on Salmonella adhesion and Salmonella-induced cytotoxicity of Caco-2 cells subjected to thermal stress. Results Thermal stress increased the cytotoxic effect of both S. Typhimurium (P = 0.0001 and nonpathogenic E. coli K12 (P = 0.004 to Caco-2 cells, and resulted in greater susceptibility of cell monolayers to S. Typhimurium adhesion (P = 0.001. Thermal stress had no significant impact on inflammatory cytokines released by Caco-2 cells, although exposure to S. Typhimurium resulted in greater than 80% increase in production of IL-6 and IL-8. Blocking S. Typhimurium with anti-ShdA antibody prior to exposure of Salmonella decreased adhesion (P = 0.01 to non-stressed and thermal-stressed Caco-2 cells. Pre-exposure of Caco-2 cells to L. rhamnosus GG significantly reduced Salmonella-induced cytotoxicity (P = 0.001 and Salmonella adhesion (P = 0.001 to Caco-2 cells during thermal stress, while L. gasseri had no effect. Conclusion Results suggest that thermal stress increases susceptibility of intestinal epithelial Caco-2 cells to Salmonella adhesion, and increases the cytotoxic effect of Salmonella during infection. Use of L. rhamnosus GG as a probiotic may reduce the severity of infection during epithelial cell stress. Mechanisms

  12. Uptake of perfluorooctanoic acid by Caco-2 cells: Involvement of organic anion transporting polypeptides.

    Science.gov (United States)

    Kimura, Osamu; Fujii, Yukiko; Haraguchi, Koichi; Kato, Yoshihisa; Ohta, Chiho; Koga, Nobuyuki; Endo, Tetsuya

    2017-08-05

    The mechanism underlying the intestinal absorption of perfluorooctanoic acid (PFOA) was investigated using Caco-2 cells. The uptake of PFOA from the apical membrane of Caco-2 cells was fast, and pH, temperature, and concentration dependent, but Na+ independent. Coincubation with sulfobromophthalein (BSP), glibenclamide, estron-3-sulfate, cyclosporine A or rifamycin SV, which are typical substrates or inhibitors of organic anion transporting polypeptides (OATPs), significantly decreased the uptake of PFOA. However, coincubation with probenecid or p-aminohippuric acid, typical substrates of organic anion transporters, did not decrease the uptake of PFOA. Furthermore, coincubation with l-lactic acid or benzoic acid, substrates of monocarboxylic acid transporters, did not decrease PFOA uptake. The relationship between the initial uptake of PFOA and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The calculated Km and uptake clearance (Vmax/Km) values for PFOA were 8.3μM and 55.0μL/mg protein/min, respectively. This clearance value was about 3-fold greater than that of the non-saturable uptake clearance (Kd: 18.1μL/mg protein/min). Lineweaver-Burk plots revealed that BSP competitively inhibits the uptake of PFOA, with a Ki value of 23.1μM. These results suggest that the uptake of PFOA from the apical membranes of Caco-2 cells could be, at least in part, mediated by OATPs along with BSP. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized form MDCK cells and mainly basolaterally from Caco-2 cells

    DEFF Research Database (Denmark)

    Vogel, L K; Suske, G; Beato, M

    1993-01-01

    and Caco-2 cells thus secrete uteroglobin in a non-sorted manner. It has, however, previously been shown that uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either...

  14. Effect of Human Breast Milk on the Expression of Proinflammatory Cytokines in Caco-2 Cells after Hypoxia/Re-Oxygenation.

    Science.gov (United States)

    Ruan, Wei-Yong; Bi, Ming-Yuan; Feng, Wei-Wei; Wang, Yu-Jun; Bu, Wei-Quan; Lu, Ling

    2016-01-01

    Neonatal necrotizing enterocolitis is a common and often fatal gastrointestinal disease, especially in premature infants. To study potential mechanisms underlying the protective effect of breast milk on neonatal necrotizing enterocolitis, we induced intestinal inflammation in a Caco-2 cell model of neonatal necrotizing enterocolitis by hypoxia/re-oxygenation to investigate whether breast milk supernatant fluid inhibited the expression of proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α. Caco-2 cells were divided into normal (control) and neonatal necrotizing enterocolitis groups. Neonatal necrotizing enterocolitis was mimicked by exposing Caco-2 cells to hypoxia/re-oxygenation. Cells were independently maintained in minimal essential medium alone, minimal essential medium containing 5% breast milk supernatant, or 5% boiled breast milk supernatant. Production of interleukin-1β, interleukin-6, and tumor necrosis factor-α was investigated in cell culture supernatants by ELISA, reverse transcription polymerase chain reaction, and immunofluorescence. Hypoxia/re-oxygenation significantly increased the expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α. In the normal group, breast milk supernatant and boiled breast milk supernatant markedly downregulated the expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α when compared with the minimal essential medium group, with the reduction in inter-leukin-1β expression being more pronounced in the breast milk group. In Caco-2 cells undergoing hypoxia/re-oxygenation, both breast milk supernatant and boiled breast milk supernatant significantly reduced the expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α, where the decrease in interleukin-1β expression was greater in the breast milk group. Breast milk supernatant fluid inhibited the expression of proinflammatory cytokines interleukin-1β, interleukin-6, and tumor

  15. Acamprosate permeability across Caco-2 cell monolayer is predominantly paracellular

    DEFF Research Database (Denmark)

    Antonescu, Irina-Elena; Steffansen, Bente

    /MS-MS. For the neutral paracellular marker [14C]-mannitol, which has a very similar molecular radius compared to acamprosate, the Papp, exp was measured in parallel. The experimental value for acamprosate was then compared to a mechanistically modelled Caco-2 apparent permeability, Papp, calc. Papp, calc...... effective permeability of acamprosate, which is fully ionized in vivo (pKa 1.83; molecular weight 181.2 g/mol), is predominantly paracellular (Ppara) or is transcellularly mediated by solute carriers (Ptrans). Aim. The overall aim was to get a better insight on the contribution of Ppara and Ptrans...... to the overall acamprosate apparent permeability. Methods. Acamprosate apparent permeability (Papp, exp) was determined across Caco-2 monolayers in the apical-to-basolateral transport direction using a buffer pH of 7.4 and several cell passages (N). Acamprosate concentrations were quantified by LC...

  16. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  17. Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii.

    Science.gov (United States)

    Sobotta, Katharina; Bonkowski, Katharina; Liebler-Tenorio, Elisabeth; Germon, Pierre; Rainard, Pascal; Hambruch, Nina; Pfarrer, Christiane; Jacobsen, Ilse D; Menge, Christian

    2017-04-12

    Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1β, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host's immune response.

  18. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

    2013-06-14

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

  19. Facilitated nanoscale delivery of insulin across intestinal membrane models.

    Science.gov (United States)

    Woitiski, Camile B; Sarmento, Bruno; Carvalho, Rui A; Neufeld, Ronald J; Veiga, Francisco

    2011-06-30

    The effect of nanoparticulate delivery system on enhancing insulin permeation through intestinal membrane was evaluated in different intestinal epithelial models using cell cultures and excised intestinal tissues. Multilayered nanoparticles were formulated by encapsulating insulin within a core consisting of alginate and dextran sulfate nucleating around calcium and binding to poloxamer, stabilized by chitosan, and subsequently coated with albumin. Insulin permeation through Caco-2 cell monolayer was enhanced 2.1-fold, facilitated by the nanoparticles compared with insulin alone, 3.7-fold through a mucus-secreting Caco-2/HT29 co-culture, and 3.9-fold through excised intestinal mucosa of Wistar rats. Correlation of Caco-2/HT29 co-culture cells with the animal-model intestinal membrane demonstrates that the mucus layer plays a significant role in determining the effectiveness of oral nanoformulations in delivering poorly absorbed drugs. Albumin was applied to the nanoparticles as outermost coat to protect insulin through shielding from proteolytic degradation. The effect of the albumin layering on insulin permeation was compared with albumin-free nanoparticles that mimic the result of albumin being enzymatically removed during gastric and intestinal transport. Results showed that albumin layering is important toward improving insulin transport across the intestinal membrane, possibly by stabilizing insulin in the intestinal conditions. Transcellular permeation was evidenced by internalization of independently labeled insulin and nanoparticles into enterocytes, in which insulin appeared to remain associated with the nanoparticles. Transcellular transport of insulin through rat intestinal mucosa may represent the predominant mechanism by which nanoparticles facilitate insulin permeation. Nanoformulations demonstrated biocompatibility with rat intestinal mucosa through determination of cell viability via monitoring of mitochondrial dehydrogenases. Insulin permeation

  20. Assesing potential effects of inulin and probiotic bacteria on Fe bioavailability from common beans (Phaseolus vulgaris L.) to Caco-2 cells

    Science.gov (United States)

    Inulin, a prebiotic, may enhance intestinal Fe absorption. Our objective was to assess the effects of supplemental inulin and two probiotic bacteria (B. infantis and L.acidophillus) on Fe availability to Caco-2 cells from common white and red beans (Phaseolus vulgaris L.). Cooked beans were mixed o...

  1. Characteristics of ceftibuten uptake into Caco-2 cells.

    Science.gov (United States)

    Muranushi, N; Horie, K; Masuda, K; Hirano, K

    1994-12-01

    The characteristics of ceftibuten uptake into Caco-2 cells grown in a collagen-coated dish were examined. Ceftibuten showed stereoselective and pH-dependent uptake. The pH-dependency of ceftibuten was more marked than that of cefaclor or cephalexin, but all three antibiotics showed maximal uptake at pH 5.5. Ceftibuten uptake was linear for the initial 1 hr and then reached a plateau. The initial uptake (15 min) was markedly reduced by the addition of 2,4-dinitrophenol or FCCP (a protonophore), or by lowering the incubation temperature. The uptake of ceftibuten into the brush-border membrane vesicles prepared from cultured Caco-2 cells showed an overshoot in the presence of an H(+)-gradient. These findings indicated that the uptake of ceftibuten was energy-dependent, especially H(+)-gradient-dependent. Uptake inhibition by various compounds was compared using Caco-2 cells. Amino acids and a tetrapeptide did not inhibit uptake, whereas di- or tri-peptides were effective inhibitors. These observations suggest that ceftibuten is taken up by a carrier-mediated transport system(s) for dipeptides. Various antibiotics differed in their ability to inhibit uptake, with cyclacillin showing maximum inhibition. Differences in the inhibitory effect may be accounted for by the heterogeneity (multiplicity) of the transport systems.

  2. Transepithelial Transport of PAMAM Dendrimers Across Isolated Human Intestinal Tissue.

    Science.gov (United States)

    Hubbard, Dallin; Enda, Michael; Bond, Tanner; Moghaddam, Seyyed Pouya Hadipour; Conarton, Josh; Scaife, Courtney; Volckmann, Eric; Ghandehari, Hamidreza

    2015-11-02

    Poly(amido amine) (PAMAM) dendrimers have shown transepithelial transport across intestinal epithelial barrier in rats and across Caco-2 cell monolayers. Caco-2 models innately lack mucous barriers, and rat isolated intestinal tissue has been shown to overestimate human permeability. This study is the first report of transport of PAMAM dendrimers across isolated human intestinal epithelium. It was observed that FITC labeled G4-NH2 and G3.5-COOH PAMAM dendrimers at 1 mM concentration do not have a statistically higher permeability compared to free FITC controls in isolated human jejunum and colonic tissues. Mannitol permeability was increased at 10 mM concentrations of G3.5-COOH and G4-NH2 dendrimers. Significant histological changes in human colonic and jejunal tissues were observed at G3.5-COOH and G4-NH2 concentrations of 10 mM implying that dose limiting toxicity may occur at similar concentrations in vivo. The permeability through human isolated intestinal tissue in this study was compared to previous rat and Caco-2 permeability data. This study implicates that PAMAM dendrimer oral drug delivery may be feasible, but it may be limited to highly potent drugs.

  3. Acamprosate has no impact on the permeability of paracellular markers across Caco-2 cells

    DEFF Research Database (Denmark)

    Antonescu, Irina; Steffansen, Bente; Neuhoff, Sibylle

    of the paracellular markers, mannitol and Lucifer Yellow (LY), was investigated. Methods: Ppara of LY and [14C]-mannitol was investigated across filter grown human epithelial colorectal adenocarcinoma (Caco-2) cell monolayers. Changes in the transepithelial electrical resistance (TEER) across the monolayers were...... evaluated. Results: LY and [14C]-mannitol Ppara values were not significantly different in presence 0.43±0.06x10-6 and 0.47±0.24x10-6 cm/s or absence 0.30±0.08x10-6 and 0.43±0.20x10-6 cm/s (Mean±SD, n=3) of acamprosate, respectively. Pre-treated cells with acamprosate for 24 or 4 hours before applying...... the [14C]-mannitol, Papp values of 0.71±0.2x10-6 and 0.51±0.17x10-6 cm/s were obtained. TEER values at the end of all experiments were in the range of 426-444 ohm*cm2. Summary/Conclusion: Acamprosate has no impact on the paracellular pathway across Caco-2 cell monolayers of LY and mannitol, or on the TEER...

  4. Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells.

    Science.gov (United States)

    Hiramatsu, Yukihiro; Yamamoto, Masatatsu; Satho, Tomomitsu; Irie, Keiichi; Kai, Akiko; Uyeda, Saori; Fukumitsu, Yuki; Toda, Akihisa; Miyata, Takeshi; Miake, Fumio; Arakawa, Takeshi; Kashige, Nobuhiro

    2014-05-10

    Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB-YVAD). YVAD is a tetrapeptide (tyrosine-valine-alanine-aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB-YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs. We constructed the rCTB-YVAD secretion vector pSCTB-YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB-YVAD was generated by transformation with pSCTB-YVAD. Both the culture supernatant of pSCTB-YVAD-transformed L. casei and purified rCTB-YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB-YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB-YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability. The rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant

  5. Establishment of an In Vitro Intestinal Epithelial Cell Culture Model of Avian Origin.

    Science.gov (United States)

    Kaiser, Annette; Willer, Thomas; Steinberg, Pablo; Rautenschlein, Silke

    2017-06-01

    The role of intestinal epithelial cells (IECs) in the physiology of the gastrointestinal tract (GIT) of chickens and pathogenesis of various diseases in chickens is still poorly understood. IECs line the GIT and represent the border between the unsterile environment and the sterile internal tissues. Bacterial, viral, fungal, or parasitic pathogens are able to invade or pass IECs under certain circumstances and cause various diseases. Pathogen-host interactions in the chicken gut are poorly understood because of the lack of suitable in vitro and ex vivo models. In this context, there is a need to optimize the cell isolation and culture conditions to be able to provide reproducible IEC cultures with defined epithelial characteristics. We compared different mechanical IEC isolation protocols and cell culture media and established a reproducible primary intestinal epithelial cell culture model from specific-pathogen-free layer-type chickens. By using isolated crypts from the duodenum of 5- to 12-wk-old birds to create the starting material, we were able to culture replicating cells between 7 and 10 days. Cells built an almost closed monolayer and showed epithelial-like characteristics, such as the expression of cytokeratin and epithelial cadherin. The primary IEC cultures described in this study represent a suitable model with which to investigate in vitro pathogen-host interactions relevant to the chicken gut.

  6. Effect of Psychoneural Factors on Intestinal Epithelial Function

    Directory of Open Access Journals (Sweden)

    M Cecilia Berin

    1997-01-01

    Full Text Available Stress has been associated with abnormal gastrointestinal function, including diarrhea and abdominal pain, and stress-associated gastric ulceration has frequently been documented. Stress can also exacerbate ongoing pathophysiology and often precedes relapses in patients with inflammatory bowel disease or irritable bowel syndrome. The relatively new field of psychoneuroimmunology is involved with the elucidation of mechanisms that explain the link between the central nervous system and immune-mediated pathophysiology. Recent progress examining the interaction among the nervous system, the immune system and the epithelium of the intestine is discussed, and the evidence for central nervous sysytem control of this interaction is examined.

  7. RGD-Dependent Epithelial Cell-Matrix Interactions in the Human Intestinal Crypt

    Directory of Open Access Journals (Sweden)

    Yannick D. Benoit

    2012-01-01

    Full Text Available Interactions between the extracellular matrix (ECM and integrin receptors trigger structural and functional bonds between the cell microenvironment and the cytoskeleton. Such connections are essential for adhesion structure integrity and are key players in regulating transduction of specific intracellular signals, which in turn regulate the organization of the cell microenvironment and, consequently, cell function. The RGD peptide-dependent integrins represent a key subgroup of ECM receptors involved in the maintenance of epithelial homeostasis. Here we review recent findings on RGD-dependent ECM-integrin interactions and their roles in human intestinal epithelial crypt cells.

  8. Intestinal Epithelial Cell Endoplasmic Reticulum Stress and Inflammatory Bowel Disease Pathogenesis: An Update Review

    Science.gov (United States)

    Ma, Xiaoshi; Dai, Zhaolai; Sun, Kaiji; Zhang, Yunchang; Chen, Jingqing; Yang, Ying; Tso, Patrick; Wu, Guoyao; Wu, Zhenlong

    2017-01-01

    The intestinal epithelial cells serve essential roles in maintaining intestinal homeostasis, which relies on appropriate endoplasmic reticulum (ER) function for proper protein folding, modification, and secretion. Exogenous or endogenous risk factors with an ability to disturb the ER function can impair the intestinal barrier function and activate inflammatory responses in the host. The last decade has witnessed considerable progress in the understanding of the functional role of ER stress and unfolded protein response (UPR) in the gut homeostasis and its significant contribution to the pathogenesis of inflammatory bowel disease (IBD). Herein, we review recent evidence supporting the viewpoint that deregulation of ER stress and UPR signaling in the intestinal epithelium, including the absorptive cells, Paneth cells, goblet cells, and enteroendocrine cells, mediates the action of genetic or environmental factors driving colitis in experimental animals and IBD patients. In addition, we highlight pharmacologic application of chaperones or small molecules that enhance protein folding and modification capacity or improve the function of the ER. These molecules represent potential therapeutic strategies in the prevention or treatment of IBD through restoring ER homeostasis in intestinal epithelial cells. PMID:29118753

  9. Intestinal Epithelial Cell Endoplasmic Reticulum Stress and Inflammatory Bowel Disease Pathogenesis: An Update Review

    Directory of Open Access Journals (Sweden)

    Xiaoshi Ma

    2017-10-01

    Full Text Available The intestinal epithelial cells serve essential roles in maintaining intestinal homeostasis, which relies on appropriate endoplasmic reticulum (ER function for proper protein folding, modification, and secretion. Exogenous or endogenous risk factors with an ability to disturb the ER function can impair the intestinal barrier function and activate inflammatory responses in the host. The last decade has witnessed considerable progress in the understanding of the functional role of ER stress and unfolded protein response (UPR in the gut homeostasis and its significant contribution to the pathogenesis of inflammatory bowel disease (IBD. Herein, we review recent evidence supporting the viewpoint that deregulation of ER stress and UPR signaling in the intestinal epithelium, including the absorptive cells, Paneth cells, goblet cells, and enteroendocrine cells, mediates the action of genetic or environmental factors driving colitis in experimental animals and IBD patients. In addition, we highlight pharmacologic application of chaperones or small molecules that enhance protein folding and modification capacity or improve the function of the ER. These molecules represent potential therapeutic strategies in the prevention or treatment of IBD through restoring ER homeostasis in intestinal epithelial cells.

  10. Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

    Science.gov (United States)

    Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

    2017-12-01

    In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5–3 µm were used and a shear stress of ~0.03 dyne cm‑2 was created by applying a low flow rate of 20 nl s‑1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

  11. Different responses of Caco-2 and MCF-7 cells to silver nanoparticles are based on highly similar mechanisms of action.

    Science.gov (United States)

    van der Zande, Meike; Undas, Anna K; Kramer, Evelien; Monopoli, Marco P; Peters, Ruud J; Garry, David; Antunes Fernandes, Elsa C; Hendriksen, Peter J; Marvin, Hans J P; Peijnenburg, Ad A; Bouwmeester, Hans

    2016-12-01

    The mode of action of silver nanoparticles (AgNPs) is suggested to be exerted through both Ag+ and AgNP dependent mechanisms. Ingestion is one of the major NP exposure routes, and potential effects are often studied using Caco-2 cells, a well-established model for the gut epithelium. MCF-7 cells are epithelial breast cancer cells with extensive well-characterized toxicogenomics profiles. In the present study, we aimed to gain a deeper understanding of the cellular molecular responses in Caco-2 and MCF-7 cells after AgNP exposure in order to evaluate whether epithelial cells derived from different tissues demonstrated similar responses. These insights could possibly reduce the size of cell panels for NP hazard identification screening purposes. AgNPs of 20, 30, 60, and 110 nm, and AgNO3 were exposed for 6 h and 24 h. AgNPs were shown to be taken up and dissolve intracellularly. Compared with MCF-7 cells, Caco-2 cells showed a higher sensitivity to AgNPs, slower gene expression kinetics and absence of NP size-dependent responses. However, on a molecular level, no significant differences were observed between the two cell types. Transcriptomic analysis showed that Ag(NP) exposure caused (oxidative) stress responses, possibly leading to cell death in both cell lines. There was no indication for effects specifically induced by AgNPs. Responses to AgNPs appeared to be induced by silver ions released from the AgNPs. In conclusion, differences in mRNA responses to AgNPs between Caco-2 and MCF-7 cells were mainly related to timing and magnitude, but not to a different underlying mechanism.

  12. Intestinal Cell Tight Junctions Limit Invasion of Candida albicans through Active Penetration and Endocytosis in the Early Stages of the Interaction of the Fungus with the Intestinal Barrier.

    Directory of Open Access Journals (Sweden)

    Marianne Goyer

    Full Text Available C. albicans is a commensal yeast of the mucous membranes in healthy humans that can also cause disseminated candidiasis, mainly originating from the digestive tract, in vulnerable patients. It is necessary to understand the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In this study, we investigated the kinetics of tight junction (TJ formation in parallel with the invasion of C. albicans into the Caco-2 intestinal cell line. Using invasiveness assays on Caco-2 cells displaying pharmacologically altered TJ (i.e. differentiated epithelial cells treated with EGTA or patulin, we were able to demonstrate that TJ protect enterocytes against invasion of C. albicans. Moreover, treatment with a pharmacological inhibitor of endocytosis decreased invasion of the fungus into Caco-2 cells displaying altered TJ, suggesting that facilitating access of the yeast to the basolateral side of intestinal cells promotes endocytosis of C. albicans in its hyphal form. These data were supported by SEM observations of differentiated Caco-2 cells displaying altered TJ, which highlighted membrane protrusions engulfing C. albicans hyphae. We furthermore demonstrated that Als3, a hypha-specific C. albicans invasin, facilitates internalization of the fungus by active penetration and induced endocytosis by differentiated Caco-2 cells displaying altered TJ. However, our observations failed to demonstrate binding of Als3 to E-cadherin as the trigger mechanism of endocytosis of C. albicans into differentiated Caco-2 cells displaying altered TJ.

  13. Four Cation-Selective Transporters Contribute to Apical Uptake and Accumulation of Metformin in Caco-2 Cell Monolayers

    Science.gov (United States)

    Han, Tianxiang (Kevin); Proctor, William R.; Costales, Chester L.; Cai, Hao; Everett, Ruth S.

    2015-01-01

    Metformin is the frontline therapy for type II diabetes mellitus. The oral bioavailability of metformin is unexpectedly high, between 40 and 60%, given its hydrophilicity and positive charge at all physiologic pH values. Previous studies in Caco-2 cell monolayers, a cellular model of the human intestinal epithelium, showed that during absorptive transport metformin is taken up into the cells via transporters in the apical (AP) membrane; however, predominant transport to the basolateral (BL) side occurs via the paracellular route because intracellular metformin cannot egress across the BL membrane. Furthermore, these studies have suggested that the AP transporters can contribute to intestinal accumulation and absorption of metformin. Transporter-specific inhibitors as well as a novel approach involving a cocktail of transporter inhibitors with overlapping selectivity were used to identify the AP transporters that mediate metformin uptake in Caco-2 cell monolayers; furthermore, the relative contributions of these transporters in metformin AP uptake were also determined. The organic cation transporter 1, plasma membrane monoamine transporter (PMAT), serotonin reuptake transporter, and choline high-affinity transporter contributed to approximately 25%, 20%, 20%, and 15%, respectively, of the AP uptake of metformin. PMAT-knockdown Caco-2 cells were constructed to confirm the contribution of PMAT in metformin AP uptake because a PMAT-selective inhibitor is not available. The identification of four intestinal transporters that contribute to AP uptake and potentially intestinal absorption of metformin is a significant novel finding that can influence our understanding of metformin pharmacology and intestinal drug-drug interactions involving this highly prescribed drug. PMID:25563903

  14. File list: Oth.Dig.50.AllAg.Caco-2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. Interaction with Intestinal Epithelial Cells Promotes an Immunosuppressive Phenotype in Lactobacillus casei

    Science.gov (United States)

    Tiittanen, Minna; Keto, Joni; Haiko, Johanna; Mättö, Jaana; Partanen, Jukka; Lähteenmäki, Kaarina

    2013-01-01

    Maintenance of the immunological tolerance and homeostasis in the gut is associated with the composition of the intestinal microbiota. We here report that cultivation of Lactobacillus casei ATCC 334 in the presence of human intestinal epithelial cells promotes functional changes in bacteria. In particular, the interaction enhanced the immunosuppressive phenotype of L. casei as demonstrated by the ability of L. casei to generate functional regulatory T cells (CD4+CD25+FoxP3+) and production of the anti-inflammatory cytokine interleukin-10 by human peripheral blood mononuclear cells. The results indicate microbe-host cross-talk that changes features of microbes, and suggest that in vitro simulation of epithelial cell interaction can reveal functional properties of gut microbes more accurately than conventional cultivation. PMID:24244309

  11. Evaluation of preparation methods for MS-based analysis of intestinal epithelial cell proteomes

    DEFF Research Database (Denmark)

    Hesselager, Marianne Overgaard; Codrea, Marius Cosmin; Bendixen, Emøke

    2015-01-01

    The gut epithelium formed between an organism and the environment plays an essential role in host–microbe interactions, yet remains one of the least characterized mammalian tissues. Especially the membrane proteins, which are critical to bacterial adhesion, are understudied, because these proteins...... are low in abundance, and large amounts of sample is needed for their preparation and for undertaking MS-based analysis. The aim of this study was to evaluate three different methods for isolation and preparation of pig intestinal epithelial cells for MS-based analysis of the proteome. Samples were...... of ease and speed of sample preparation, as well as protein recovery. In comparison, more gentle methods where intestinal epithelial cells are harvested by shaking are more time consuming, result in lower protein yield, and are prone to increased technical variation due to multiple steps involved....

  12. Probiotic Bifidobacterium species stimulate human SLC26A3 gene function and expression in intestinal epithelial cells.

    Science.gov (United States)

    Kumar, Anoop; Hecht, Cameron; Priyamvada, Shubha; Anbazhagan, Arivarasu N; Alakkam, Anas; Borthakur, Alip; Alrefai, Waddah A; Gill, Ravinder K; Dudeja, Pradeep K

    2014-12-15

    SLC26A3, or downregulated in adenoma (DRA), plays a major role in mediating Cl(-) absorption in the mammalian intestine. Disturbances in DRA function and expression have been implicated in intestinal disorders such as congenital Cl(-) diarrhea and gut inflammation. We previously showed that an increase in DRA function and expression by Lactobacillus acidophilus and its culture supernatant (CS) might underlie antidiarrheal effects of this probiotic strain. However, the effects of Bifidobacterium species, important inhabitants of the human colon, on intestinal Cl(-)/HCO3 (-) exchange activity are not known. Our current results demonstrate that CS derived from Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum increased anion exchange activity in Caco-2 cells (∼1.8- to 2.4-fold). Consistent with the increase in DRA function, CS also increased the protein, as well as the mRNA, level of DRA (but not putative anion transporter 1). CS of all three Bifidobacterium sp. increased DRA promoter activity (-1,183/+114 bp) in Caco-2 cells (1.5- to 1.8-fold). Furthermore, the increase in DRA mRNA expression by CS of B. breve and B. infantis was blocked in the presence of the transcription inhibitor actinomycin D (5 μM) and the ERK1/2 MAPK pathway inhibitor U0126 (10 μM). Administration of live B. breve, B. infantis, and B. bifidum by oral gavage to mice for 24 h increased DRA mRNA and protein levels in the colon. These data demonstrate an upregulation of DRA via activation of the ERK1/2 pathway that may underlie potential antidiarrheal effects of Bifidobacterium sp.

  13. Metabolic regulation of intestinal epithelial barrier during inflammation.

    Science.gov (United States)

    Colgan, Sean P; Curtis, Valerie F; Lanis, Jordi M; Glover, Louise E

    2015-01-01

    The gastrointestinal mucosa has proven to be an interesting tissue for which to investigate disease-related metabolism. In this review, we outline some evidence that implicates metabolic signaling as important features of barrier in the healthy and disease. Studies from cultured cell systems, animal models and human patients have revealed that metabolites generated within the inflammatory microenvironment are central to barrier regulation. These studies have revealed a prominent role for hypoxia and hypoxia-inducible factor (HIF) at key steps in adenine nucleotide metabolism and within the creatine kinase pathway. Results from animal models of intestinal inflammation have demonstrated an almost uniformly beneficial influence of HIF stabilization on disease outcomes and barrier function. Studies underway to elucidate the contribution of immune responses will provide additional insight into how metabolic changes contribute to the complexity of the gastrointestinal tract and how such information might be harnessed for therapeutic benefit.

  14. Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant.

    Science.gov (United States)

    Bu, Pengli; Narayanan, Silpa; Dalrymple, Damon; Cheng, Xingguo; Serajuddin, Abu T M

    2016-08-25

    Caco-2 cells are used extensively for in vitro prediction of intestinal drug absorption. However, toxicity of excipients and formulations used can artificially increase drug permeation by damaging cell monolayers, thus providing misleading results. The present study aimed to investigate cytotoxicity of common lipid-based excipients and formulations on Caco-2 cells. Medium-chain monoglycerides alone or in mixture with the surfactant Cremophor EL, with and without a medium-chain triglyceride, were prepared and incubated with Caco-2 cells from a series of culture stages with varying maturity. Cell viability was evaluated and cell membrane integrity assessed. Cytotoxicity of lipid-based formulations was influenced by the maturity of Caco-2 cells and formulation composition. One-day culture was most sensitive to lipids. When cultured for 5days, viability of Caco-2 cells was significantly improved. The 21-day Caco-2 monolayers maintained the highest survival rate. Microemulsion formulations exhibited significantly less cytotoxicity than neat lipids or surfactant at all stages of cell maturity, and microemulsions containing 1:1 mixtures of monoglyceride and triglyceride appeared to be best tolerated among all the formulations tested. Mechanistically, the observed cytotoxicity was partially due to lipid-induced rupture of cell membrane. Microemulsions of lipid-surfactant mixtures have less cytotoxicity than lipid alone. Maturity of Caco-2 cells renders significant resistance to cytotoxicity, and monolayers with 21-day maturity are more relevant to in vivo conditions and appear to be a more accurate in vitro model for cytotoxicity assessment. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Cell cycle arrest and apoptosis induced by enteroaggregative Escherichia coli in cultured human intestinal epithelial cells.

    Science.gov (United States)

    Priya, Anshu; Kaur, Kiranjeet; Bhattacharyya, Shalmoli; Chakraborti, Anuradha; Ghosh, Sujata

    2017-03-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen causing diarrhoeal diseases in multiple epidemiological and clinical s