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Sample records for caco-2 intestinal epithelial

  1. Milk Modulates Campylobacter Invasion into Caco-2 Intestinal Epithelial Cells.

    Science.gov (United States)

    Louwen, Rogier; van Neerven, R J Joost

    2015-09-01

    Raw milk is a recognized source of Campylobacter outbreaks, but pasteurization is an effective way to eliminate the causative agent of Campylobacteriosis. Whereas breastfeeding is protective against infectious diseases, consumption of formula milk is thought to be not. However, in relation to Campylobacter, such data is currently unavailable. Although both pasteurized and formula milk are pathogen free and prepared in a quality controlled manner, the effect they have on the virulence of Campylobacter species is unknown. Here, we studied the effect of cow, goat, horse, and formula milk on Campylobacter invasion into intestinal epithelial Caco-2 cells, a pathogenic feature of this bacterial species, using a gentamicin exclusion invasion assay. We found that all milk products modulated the invasion of Campylobacter species into the Caco-2 cells in a dose-dependent manner. Control experiments showed that the milks were not toxic for the Caco-2 cells and that the effect on invasion is caused by heat labile (e.g., milk proteins) or heat stable (e.g., sugar/lipids) components depending on the Campylobacter species studied. This in vitro study shows for the first time that pasteurized and formula milk affect the invasion of Campylobacter. We recommend a prospective study to examine whether pasteurized and formula milk affect Campylobacteriosis. PMID:26495128

  2. TNFalpha regulates sugar transporters in the human intestinal epithelial cell line Caco-2

    OpenAIRE

    Barrenetxe, J; Barber, A; Lostao, M P; Rodriguez-Yoldi, M.J. (M.J.); Gascon, S. (S.); Sanchez, O.

    2013-01-01

    PURPOSE: During intestinal inflammation TNFα levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNFα inhibits galactose, fructose and leucine intestinal absorption in animal models. In continuation with our work, the purpose of the present study was to investigate in the human intestinal epithelial cell line Caco-2, the effect of TNFα on sugar transport and to identify the intracellular mechanisms involved. METHODS: ...

  3. Uptake of codeine into intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells.

    Science.gov (United States)

    Fischer, Wiebke; Bernhagen, Jennifer; Neubert, Reinhard H H; Brandsch, Matthias

    2010-09-11

    Orally administered codeine has to permeate both the intestinal and the blood-brain barrier in order to act as analgesic and cough suppressant. In this study we characterized the uptake of codeine at intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells. At both cell types, uptake of [(3)H]codeine was independent of an inwardly directed Na(+) gradient. Uptake was, however, strongly stimulated by an outwardly directed H(+) gradient and inhibited by the protonophore FCCP. [(3)H]Codeine uptake into Caco-2 cells was strongly temperature dependent. In the presence of excess amounts of unlabeled codeine, the uptake was inhibited by up to 87% (Caco-2) or 94% (RBE4), respectively. Synthetic opioids and some non-opioid organic cations like propranolol, pyrilamine and quinidine potently inhibited [(3)H]codeine uptake. Several prototype substrates of known transporters for amino acids, neurotransmitters and organic cations were ineffective. Our data are consistent with a hypothetic saturable, H(+)-dependent (antiport) mechanism not yet identified on a molecular level. The pH dependence of codeine uptake and its intracellular accumulation can partially also be explained by a model comprising diffusional membrane permeation of unionized species of codeine followed by codeine sequestration into acidic vesicles and distribution into cellular lipids. PMID:20510359

  4. Eicosapentaenoic acid enhances heat stress-impaired intestinal epithelial barrier function in Caco-2 cells.

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    Guizhen Xiao

    Full Text Available OBJECTIVE: Dysfunction of the intestinal epithelial tight junction (TJ barrier is known to have an important etiologic role in the pathophysiology of heat stroke. N-3 polyunsaturated fatty acids (PUFAs, including eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA, play a role in maintaining and protecting the TJ structure and function. This study is aimed at investigating whether n-3 PUFAs could alleviate heat stress-induced dysfunction of intestinal tight junction. METHODS: Human intestinal epithelial Caco-2 cells were pre-incubated with EPA, DHA or arachidonic acid (AA and then exposed to heat stress. Transepithelial electrical resistance (TEER and Horseradish Peroxidase (HRP permeability were measured to analyze barrier integrity. Levels of TJ proteins, including occludin, ZO-1 and claudin-2, were analyzed by Western blot and localized by immunofluorescence microscopy. Messenger RNA levels were determined by quantitative real time polymerase chain reaction (Q-PCR. TJ morphology was observed by transmission electron microscopy. RESULTS: EPA effectively attenuated the decrease in TEER and impairment of intestinal permeability in HRP flux induced by heat exposure. EPA significantly elevated the expression of occludin and ZO-1, while DHA was less effective and AA was not at all effective. The distortion and redistribution of TJ proteins, and disruption of morphology were also effectively prevented by pretreatment with EPA. CONCLUSION: This study indicates for the first time that EPA is more potent than DHA in protecting against heat-induced permeability dysfunction and epithelial barrier damage of tight junction.

  5. 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. PMID:26923511

  6. Celiac anti-type 2 transglutaminase antibodies induce phosphoproteome modification in intestinal epithelial Caco-2 cells.

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    Gaetana Paolella

    Full Text Available BACKGROUND: Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2 activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. METHODS AND PRINCIPAL FINDINGS: We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins, three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. CONCLUSIONS: Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here

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

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    Xiang, Jinmei; Guo, Rui; Wan, Chunyun; Wu, Liming; Yang, Shijin; Guo, Dingzong

    2016-01-01

    Stanniocalcin-1 (STC1) is a calcium and phosphate regulatory hormone. However, the exact molecular mechanisms underlying how STC1 affects Ca(2+) 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. PMID:27409607

  8. Eicosapentaenoic Acid Enhances Heat Stress-Impaired Intestinal Epithelial Barrier Function in Caco-2 Cells

    OpenAIRE

    Guizhen Xiao; Liqun Tang; Fangfang Yuan; Wei Zhu; Shaoheng Zhang; Zhifeng Liu; Yan Geng; Xiaowen Qiu; Yali Zhang; Lei Su

    2013-01-01

    OBJECTIVE: Dysfunction of the intestinal epithelial tight junction (TJ) barrier is known to have an important etiologic role in the pathophysiology of heat stroke. N-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), play a role in maintaining and protecting the TJ structure and function. This study is aimed at investigating whether n-3 PUFAs could alleviate heat stress-induced dysfunction of intestinal tight junction. METHODS: Human i...

  9. 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.

  10. Cytokine modulation (IL-6, IL-8, IL-10) by human breast milk lipids on intestinal epithelial cells (Caco-2).

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    Barrera, Girolamo J; Sánchez, Gabriela

    2016-08-01

    Human breast milk is the best form of nourishment for infants during the first year of life. It is composed by a complex mixture of carbohydrates, proteins and fats. Breast milk provides nutrients and bioactive factors that themselves modulate maturation and development of the gastrointestinal tract. Many studies have shown that it provides protection against gastrointestinal tract inflammation. In this sense, this study aimed to evaluate the effect of human breast milk lipids on epithelial intestinal cells (Caco-2) cytokine regulation and the fatty acid transporter protein (FATP) involved in this process. Caco-2 cells were cultivated and stimulated with different concentration of human milk lipids from healthy human mothers (18-30-year-olds) or single commercial lipids for 48 h. We measured the concentrations and mRNA levels of IL-6, IL-8 and IL-10 cytokines by immunoassay (ELISA) and quantitative-PCR (qRT-PCR) technique, respectively. We observed a two to three times decrease in pro-inflammatory cytokine levels (p < 0.01) as well as an increase in anti-inflammatory IL-10 levels in cells stimulated with increasing concentrations of breast milk lipids. These results suggest that human breast milk lipids could have an important role on the cytokine modulation in the newborn bowel. PMID:26441050

  11. Transport of quercetin di-sodium salt in the human intestinal epithelial Caco-2 cell monolayer 139.

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    Milane, H A; Al Ahmad, A; Naitchabane, M; Vandamme, T F; Jung, L; Ubeaud, G

    2007-01-01

    Quercetin di-sodium salt (QDS), a water-soluble derivative of quercetin (Q), is a potent free radical scavenger. The aim of this study was to examine the in vitro intestinal transport of QDS compared to that of Q using the Caco-2 human intestinal epithelial cell line. The apical (A) to basolateral (B) transport of QDS was found to be higher than the B to A transport of this compound. This polarized transport involved the presence of a carrier protein system. The involvement of the sodium/glucose transporter-1 (SGLT-1) was shown by using phloridzin, a selective inhibitor of this conveyor system. However, the transport of Q was not affected by this inhibitor. Moreover, the influx of QDS was pH-sensitive and decreased at pH 5.5 compared with that observed at pH 7.4 and 6.5. The permeability of QDS was 10-fold higher than that of Q. This could be explained by the involvement of SLGT-1 and the absence of an active efflux pump in the absorption of QDS in comparison with Q. This finding was supported by comparing the solubility of Q with that of QDS. This study indicates that both the higher solubility of QDS and its dependence on the SGLT-1 transport system resulted in more efficient permeability compared to Q. PMID:18062406

  12. 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

    resulting in a high throughput experimental method of identifying direct targets of specific transcription factors. The method was applied to CDX2, leading to the identification of the direct binding of CDX2 to several known and novel target genes in the intestinal cell. Examination of the transcript levels...

  13. 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 generat...

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

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    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.

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

    OpenAIRE

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

    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 ...

  16. 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

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    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. PMID:27014210

  17. 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

    OpenAIRE

    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 displayed similar germination efficiencies in brain heart infusion broth.

  18. Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

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    W. Cui

    2010-04-01

    Full Text Available The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

  19. 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

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

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    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.

  2. 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

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

    International Nuclear Information System (INIS)

    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 LD50 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 than

  4. 一种新的小檗碱衍生物CPU-86017在人小肠上皮细胞(Caco-2细胞)中的转运与摄取特性%Transport and uptake characteristics of a new derivative of berberine (CPU-86017) by human intestinal epithelial cell line: Caco-2

    Institute of Scientific and Technical Information of China (English)

    杨海涛; 王广基

    2003-01-01

    AIM: The characteristics of transepithelial transport and uptake of CPU-86017 {[7-(4-chlorbenzyl)-7,8,13,13α tetrahydroberberine chloride, CTHB] }, a new antiarrhythmia agent and a new derivative of berberine, were investi gated on epithelial cell line (Caco-2) to further understand the absorption mechanism of berberine and its derivatives. METHODS: Caco-2 cell was used. RESULTS: 1) The permeability coefficient from the apical (AP) to basolateral (BL) of CPU-86017 was approximately 5 times higher than that from BL-to-AP transport. The effects of a P-glycoprotein (P-gp) inhibitor-cyclosporin A, some surfactants, and lower pH on the transepithelial transport of CPU-86017 were also observed. Cyclosporine A at 7.5 mg/L had no effect on the transepithelial electrical resistance (TEER); an about 4-fold enhancement on the transepithlial transport of CPU-86017 was observed. Some surfac tants (sodium citrate, sodium deoxycholate, and sodium dodecyl sulfate) at 100 μmol/L and low pH (pH=6.0) induced a reversible decrease of TEER; enhancements of the transepithelial transport of CPU-86017 were also observed with some surfactants; 2) In the process of uptake of CPU-86017, the initial uptake rates of CPU-86017 were saturable with a Vmax of (250+39) μg.min-1.g-1 (protein) and Km of (0.90+0.12) mmol/L. This process was enhanced by cyclosporine A (7.5 mg/L) with a Vmax of (588+49) μg.min-1.g-1 (protein) and Km (0.42+0.08) mmol/L. CONCLUSION: Some surfactants and P-gp inhibitors can be considered as enhancers of its transepithelial trans port and uptake.

  5. Vitamin A metabolism in the human intestinal Caco-2 cell line

    International Nuclear Information System (INIS)

    The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activites, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with β-carotene, retinyl acetate, or retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte. These data suggest the LRAT may be the physiologically important enzyme for the esterification of retinol in Caco-2 cells

  6. Elucidation of the Intestinal Absorption Mechanism of Celastrol Using the Caco-2 Cell Transwell Model.

    Science.gov (United States)

    Li, Hong; Li, Jie; Liu, Lu; Zhang, Yichuan; Luo, Yili; Zhang, Xiaoli; Yang, Peng; Zhang, Manna; Yu, Weifeng; Qu, Shen

    2016-08-01

    Celastrol, a triterpenoid isolated from stem (caulis) of Celastrus orbiculatus Thunb. (Celastraceae), has been known to have various pharmacological effects, including anti-inflammatory, anticancer, and antioxidant activities. However, the mechanism of the intestinal absorption of celastrol is unknown. The aim of this study was to investigate the intestinal absorption of celastrol using the Caco-2 cell transwell model. First, the bidirectional transport of celastrol in Caco-2 cell monolayers was observed. Then, the effects of time, concentration, temperature, paracellular pathway, and efflux transport inhibition on the transport of celastrol across the Caco-2 cell monolayers were investigated. The P-glycoprotein inhibitor verapamil and cyclosporin A, the multidrug resistance protein 2 inhibitor MK571, and the breast cancer resistance protein inhibitor reserpine were used. Additionally, the effects of celastrol on the activity of P-glycoprotein were evaluated using the rhodamine 123 uptake assay. In this study, we found that the intestinal transport of celastrol was a time- and concentration-dependent active transport. The paracellular pathway was not involved in the transport of celastrol, and the efflux of celastrol was energy dependent. The results indicated that celastrol is a substrate of P-glycoprotein but not multidrug resistance protein 2 or the breast cancer resistance protein. In addition, celastrol could not affect the uptake of rhodamine 123 in Caco-2 cells, which indicated that celastrol could not inhibit or induce the activity of P-glycoprotein. PMID:27159672

  7. Factors derived from Escherichia coli Nissle 1917, grown in different growth media, enhance cell death in a model of 5-fluorouracil-induced Caco-2 intestinal epithelial cell damage.

    Science.gov (United States)

    Wang, Hanru; Bastian, Susan E P; Lawrence, Andrew; Howarth, Gordon S

    2015-01-01

    We evaluated supernatants (SNs) from Escherichia coli Nissle 1917 (EcN) grown in commonly used growth media for their capacity to affect the viability of Caco-2 colon cancer cells in the presence and absence of 5-Fluorouracil (5-FU) chemotherapy. EcN was grown in Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17). Human Caco-2 colon cancer cells were treated with DMEM (control), growth media alone (LB, TSB, MRS, and M17) or EcN SNs derived from these 4 media, in the presence and absence of 5-FU. Cell viability, reactive oxygen species (ROS), and cell monolayer permeability were determined. EcN SN in LB medium reduced Caco-2 cell viability significantly, to 51% at 48 h. The combination of this EcN SN and 5-FU further reduced cell viability to 37% at 48 h, compared to 5-FU control. MRS broth and EcN SN in MRS, together with 5-FU, generated significantly lower levels of ROS compared to 5-FU control. However, all 5-FU treatments significantly disrupted the Caco-2 cell barrier compared to control; with no significant differences observed among any of the 5-FU treatments. EcN SNs (LB+) was most effective at decreasing the viability of Caco-2 cells. This could indicate a potential role for this EcN SN in chemoprevention for colon cancer. PMID:25625670

  8. Polarity of fatty acid uptake and metabolism in a human intestinal cell line (CACO-2)

    International Nuclear Information System (INIS)

    Free fatty acids (ffa) can enter the intestinal cell via the apical (AP) or basolateral (BL) membrane. The authors are using the Caco-2 intestinal cell line to examine the polarity of ffa uptake and metabolism in the enterocyte. Cells are grown on permeable polycarbonate Transwell filters in order to obtain access to both AP and BL compartments. Differentiated Caco-2 cells form tight polarized monolayers which express small intestine-specific enzymes and are impermeable to the fluid phase marker Lucifer Yellow. Submicellar concentrations of 3H-palmitic acid (2uM) were added to AP or BL sides of Caco-2 monolayers at 37 degrees C and cells were incubated for various times between 2 and 120 minutes. Total AP and BL uptake is similar; however, when relative membrane surface areas are accounted for, AP uptake is about 2-fold higher. The metabolism of AP and BL ffa is not significantly different: triacylglycerol and phosphatidylcholine account for most of the metabolites (32±4 and 24±2% respectively at 5 minutes). Little ffa oxidation is observed. Preincubation with albumin-bound 2-monoolein (100uM) and palmitate (50uM) increases the level of TG metabolites. The results suggest that in this cell line the uptake of AP ffa may be greater than BL ffa, but that AP (dietary) ffa and BL (plasma) ffa are metabolized similarly

  9. Polarity of fatty acid uptake and metabolism in a human intestinal cell line (CACO-2)

    Energy Technology Data Exchange (ETDEWEB)

    Trotter, P.J.; Storch, J. (Harvard School of Public Health, Boston, MA (United States))

    1990-02-26

    Free fatty acids (ffa) can enter the intestinal cell via the apical (AP) or basolateral (BL) membrane. The authors are using the Caco-2 intestinal cell line to examine the polarity of ffa uptake and metabolism in the enterocyte. Cells are grown on permeable polycarbonate Transwell filters in order to obtain access to both AP and BL compartments. Differentiated Caco-2 cells form tight polarized monolayers which express small intestine-specific enzymes and are impermeable to the fluid phase marker Lucifer Yellow. Submicellar concentrations of {sup 3}H-palmitic acid (2uM) were added to AP or BL sides of Caco-2 monolayers at 37{degrees}C and cells were incubated for various times between 2 and 120 minutes. Total AP and BL uptake is similar; however, when relative membrane surface areas are accounted for, AP uptake is about 2-fold higher. The metabolism of AP and BL ffa is not significantly different: triacylglycerol and phosphatidylcholine account for most of the metabolites (32{plus minus}4 and 24{plus minus}2% respectively at 5 minutes). Little ffa oxidation is observed. Preincubation with albumin-bound 2-monoolein (100uM) and palmitate (50uM) increases the level of TG metabolites. The results suggest that in this cell line the uptake of AP ffa may be greater than BL ffa, but that AP (dietary) ffa and BL (plasma) ffa are metabolized similarly.

  10. Enhanced uptake and transport of (+-catechin and (--epigallocatechin gallate in niosomal formulation by human intestinal Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Song Q

    2014-05-01

    Full Text Available Qinxin Song,1–3 Danhui Li,3 Yongzhi Zhou,3 Jie Yang,1 Wanqi Yang,1 Guohua Zhou,2 Jingyuan Wen31Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, School of Pharmacy, China Pharmaceutical University, 2Department of Pharmacology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, People’s Republic of China; 3School of Pharmacy, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New ZealandAbstract: The aim of this study was to evaluate (+-catechin and (−-epigallocatechin gallate (EGCG cellular uptake and transport across human intestinal Caco-2 cell monolayer in both the absence and presence of niosomal carrier in variable conditions. The effect of free drugs and drug-loaded niosomes on the growth of Caco-2 cells was studied. The effects of time, temperature, and concentration on drug cellular uptake in the absence or presence of its niosomal delivery systems were investigated. The intestinal epithelial membrane transport of the drug-loaded niosomes was examined using the monolayer of the human Caco-2 cells. The kinetics of transport, and the effect of temperature, adenosine triphosphate inhibitor, permeability glycoprotein inhibitor, multidrug resistance-associated protein 2 inhibitor, and the absorption enhancer on transport mechanism were investigated. It was found that the uptake of catechin, EGCG, and their niosomes by Caco-2 cells was 1.22±0.16, 0.90±0.14, 3.25±0.37, and 1.92±0.22 µg/mg protein, respectively (n=3. The apparent permeability coefficient values of catechin, EGCG, and their niosomes were 1.68±0.16, 0.88±0.09, 2.39±0.31, and 1.42±0.24 cm/second (n=3 at 37°C, respectively. The transport was temperature- and energy-dependent. The inhibitors of permeability glycoprotein and multidrug resistance-associated protein 2 and the absorption enhancer significantly enhanced the uptake amount. Compared with the free drugs, niosomal formulation

  11. Bovine and soybean milk bioactive compounds: Effects on inflammatory response of human intestinal Caco-2 cells.

    Science.gov (United States)

    Calvello, Rosa; Aresta, Antonella; Trapani, Adriana; Zambonin, Carlo; Cianciulli, Antonia; Salvatore, Rosaria; Clodoveo, Maria Lisa; Corbo, Filomena; Franchini, Carlo; Panaro, Maria Antonietta

    2016-11-01

    In this study the effects of commercial bovine and soybean milks and their bioactive compounds, namely genistein, daidzein and equol, on the inflammatory responses induced by lipopolysaccharide (LPS) treatment of human intestinal Caco-2 cells were examined, in terms of nitric oxide (NO) release and inducible nitric oxide synthetase (iNOS) expression. Both milks and their bioactive compounds significantly inhibited, dose-dependently, the expression of iNOS mRNA and protein, resulting in a decreased NO production. The NF-κB activation in LPS-stimulated intestinal cells was also examined. In all cases we observed that cell pre-treatment before LPS activation inhibited the IkB phosphorylation. Accordingly, quantification of bioactive compounds by solid phase microextraction coupled with liquid chromatography has shown that they were absorbed, metabolized and released by Caco-2 cells in culture media. In conclusion, we demonstrated that milks and compounds tested are able to reduce LPS-induced inflammatory responses from intestinal cells, interfering with NF-kB dependent molecular mechanisms. PMID:27211648

  12. Effect of linear alkylbenzene sulfonate (LAS) on human intestinal Caco-2 cells at non cytotoxic concentrations.

    Science.gov (United States)

    Bradai, Mohamed; Han, Junkyu; Omri, Abdelfatteh El; Funamizu, Naoyuki; Sayadi, Sami; Isoda, Hiroko

    2016-08-01

    Linear alkylbenzene sulfonate (LAS) is a cytotoxic synthetic anionic surfactant widely present in the environment due to its large-scale production and intensive use in the detergency field. In this study, we investigated the effect of LAS (CAS No. 25155-30-0) at non cytotoxic concentrations on human intestinal Caco-2 cells using different in vitro bioassays. As results, LAS increased Caco-2 cell proliferation at concentrations ranging from 1 to 15 ppm, more significantly for shorter exposure time (24 h), confirmed using flow cytometry and trypan blue exclusion methods. Moreover, proteomics analysis revealed that this effect was associated with an over-expression of elongation factor 2 and dipeptidyl peptidase 3, and a down-regulation of 14-3-3 protein theta, confirmed at mRNA level using real-time PCR. These findings suggest that LAS at non cytotoxic concentrations, similar to those observed at wastewater treatment plants outlets, increases the growth rate of colon cancer cells, raising thereby its tumor promotion effect potential. PMID:25999174

  13. Characterization of tight junction disruption and immune response modulation in a miniaturized Caco-2/U937 coculture-based in vitro model of the human intestinal barrier.

    Science.gov (United States)

    Ramadan, Qasem; Jing, Lin

    2016-02-01

    A microfluidic-based dynamic in vitro model of the human intestinal barrier has been constructed and characterized. The intestinal epithelial monolayer was mimicked by culturing caco-2 cells on a porous membrane in a double-layered microfluidic chip and interfaced with a co-culture of U937 as a model of immune responsive cells. The physiological flow was also mimicked by a continuous perfusion of culture media from the apical and basolateral side of the porous membrane. This dynamic "in vivo-like" environment maintains a continuous supply of cell nutrient and waste removal and create mechanical shear stress within the physiological ranges which promotes uniform cell growth and tight junction formation. The monolayer permeability to soluble ion changes after treating with LPS, and TNF α as indicated by the reduction of the TEER value. In addition, the immune competent caco-2/U937-based model allowed the investigating the role of the epithelial layer as a protection barrier to biological hazards as indicated by the suppressing of the pro-inflammatory cytokine expression. PMID:26809386

  14. Intestinal absorption of forsythoside A in in situ single-pass intestinal perfusion and in vitro Caco-2 cell models

    Institute of Scientific and Technical Information of China (English)

    Wei ZHOU; Liu-qing DI; Juan WANG; Jin-jun SHAN; Shi-jia LIU; Wen-zheng JU; Bao-chang CAI

    2012-01-01

    Aim:To investigate the mechanisms underlying the intestinal absorption of the major bioactive component forsythoside A (FTA) extracted from Forsythiae fructus.Methods:An in vitro Caco-2 cell model and a single-pass intestinal perfusion in situ model in SD rats were used.Results:In the in vitro Caco-2 cell model,the mean apparent permeability value (Papp-value) was 4.15x 107 cm/s in the apical-tobasolateral (AP-BL) direction.At the concentrations of 2.6-10.4 μg/mL,the efflux ratio of FTA in the bi-directional transport experiments was approximately 1.00.After the transport,>96% of the apically loaded FTA was retained on the apical side,while >97% of the basolaterally loaded FTA was retained on the basolateral side.The Papp-values of FTA were inversely correlated with the transepithelial electrical resistance.The paracellular permeability enhancers sodium caprate and EDTA,the P-gp inhibitor verapamil and the multidrug resistance related protein (MRP) inhibitors cyclosporine and MK571 could concentration-dependently increase the Papp-values,while the uptake (OATP) transporter inhibitors diclofenac sodium and indomethacin could concentration-dependently decrease the Papp-values.The intake transporter SGLT1 inhibitor mannitol did not cause significant change in the Papp-values.In the in situ intestinal perfusion model,both the absorption rate constant (Ka) and the effective permeability (Peff-values) following perfusion of FTA 2.6,5.2,and 10.4 μg/mL via the duodenum,jejunum and ileum had no significant difference,although the values were slightly higher for the duodenum as compared to those in the jejunum and ileum.The low,medium and high concentrations of verapamil caused the largest increase in the Peff-values for duodenum,jejunum and ileum,respectively.Sodium caprate,EDTA and cyclosporine resulted in concentration-dependent increase in the Peff-values.Diclofenac sodium and indomethacin caused concentration-dependent decrease in the Peff-values.Mannitol did

  15. Functional alterations induced by the food contaminant furazolidone on the human tumoral intestinal cell line Caco-2.

    Science.gov (United States)

    Vincentini, O; De Angelis, I; Stammati, A; Zucco, F

    1993-07-01

    Caco-2 cells, which are derived from a human colon carcinoma and are able to differentiate in culture, have been used to study the effect of furazolidone (FZ), a chemical belonging to the nitrofuran family which is frequently used for the prevention of animal infections. Its potentially toxic residues could remain in some food products of animal origin and affect human health. Toxicity has been measured by different parameters, either in undifferentiated cells (day 7 of culture), or on differentiated cells (day 21 of culture). Our results indicate that FZ may seriously affect the proliferating portion of the intestinal mucosa, while the differentiated cells appear to be more resistant. However, the slight effect recorded on the aspecific and specific functions of the differentiated cells may suggest that the specialized portion of the intestine can also be compromised by the drug. Caco 2 cells seem a good model for a deeper investigation of the mechanism involved in the toxic action of FZ. PMID:20732223

  16. Transport of hop bitter acids across intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Cattoor, Ko; Bracke, Marc; Deforce, Dieter; De Keukeleire, Denis; Heyerick, Arne

    2010-04-14

    Several health-beneficial properties of hop bitter acids have been reported (inhibition of bone resorption and anticarcinogenic and anti-inflammatory activities); however, scientific data on the bioavailability of these compounds are lacking. As a first approach to study the bioavailability, the epithelial transport of hop alpha- and beta-acids across Caco-2 monolayers was investigated. Hop acids were added either to the apical or to the basolateral chamber and, at various time points, amounts transported to the receiving compartment were determined. The monolayer integrity control was performed by using marker compounds (atenolol and propranolol), transepithelial electrical resistance (TEER) measurement, and determination of the fluorescein efflux. The TEER and fluorescein efflux confirmed the preservation of the monolayer integrity. The membrane permeability of the alpha-acids (apparent permeability coefficients for apical to basolateral transport (P(appAB)) ranged from 14 x 10(-6) to 41 x 10(-6) cm/s) was determined to be substantially higher than that of the beta-acids (P(appAB) values ranging from 0.9 x 10(-6) to 2.1 x 10(-6) cm/s). Notably, the beta-acids exhibited significantly different bidirectional P(app) values with efflux ratios around 10. The involvement of carrier-mediated transport for beta-acids (active efflux pathway by P-gp, BCRP, and/or MRP-2 type efflux pumps) could be confirmed by transport experiments with specific inhibitors (verapamil and indomethacin). It appears that alpha-acids are efficiently absorbed, whereas the permeability of beta-acids is low. Limiting factors in the absorption of beta-acids could involve P-gp and MRP-2 type efflux transporters and phase II metabolism. PMID:20329731

  17. Human intestinal absorption of imidacloprid with Caco-2 cells as enterocyte model

    International Nuclear Information System (INIS)

    In order to assess the risk to mammals of a chronic exposure to imidacloprid (IMI), we investigated its absorption with the human intestinal Caco-2 cell line. Measurements of transepithelial transport revealed an apparent permeability coefficient of 21.6 x 10-6 ± 3.2 x 10-6 cm/s reflecting a 100% absorption. The comparison of apical to basal (A-B) and basal to apical (B-A) transports showed that the monolayer presents a basal to apical polarized transport. Studies of apical uptake demonstrated that the transport was concentration-dependent and not saturable from 5 to 200 μM. Arrhenius plot analysis revealed two apparent activation energies, Ea(4-12deg.C) = 63.8 kJ/mol and Ea(12-37deg.C) 18.2 kJ/mol, suggesting two temperature-dependent processes. IMI uptake was equivalent when it was performed at pH 6.0 or 7.4. Depletion of Na+ from the transport buffer did not affect the uptake, indicating that a sodium-dependent transporter was not involved. Decrease of uptake with sodium-azide or after cell surface trypsin (Ti) treatment suggested the involvement of a trypsin-sensitive ATP-dependent transporter. Investigations on apical efflux demonstrated that initial velocities paralleled the increase of loading concentrations. A cell surface trypsin treatment did not affect the apical efflux. The lack of effect when the efflux was performed against an IMI concentration gradient suggested that an energy-dependent transporter was involved. However, the inhibition of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRP) by taxol, vincristine, and daunorubicine had no effect on IMI intracellular accumulation suggesting the involvement of transporters distinct from classical ATP binding cassette transport (ABC-transport) systems. All results suggest that IMI is strongly absorbed in vivo by inward and outward active transporters

  18. Iron depletion suppresses mTORC1-directed signalling in intestinal Caco-2 cells via induction of REDD1

    Science.gov (United States)

    Watson, Ailsa; Lipina, Christopher; McArdle, Harry J.; Taylor, Peter M.; Hundal, Harinder S.

    2016-01-01

    Iron is an indispensable micronutrient that regulates many aspects of cell function, including growth and proliferation. These processes are critically dependent upon signalling via the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Herein, we test whether iron depletion induced by cell incubation with the iron chelator, deferoxamine (DFO), mediates its effects on cell growth through mTORC1-directed signalling and protein synthesis. We have used Caco-2 cells, a well-established in vitro model of human intestinal epithelia. Iron depletion increased expression of iron-regulated proteins (TfR, transferrin receptor and DMT1, divalent metal transporter, as predicted, but it also promoted a marked reduction in growth and proliferation of Caco-2 cells. This was strongly associated with suppressed mTORC1 signalling, as judged by reduced phosphorylation of mTOR substrates, S6K1 and 4E-BP1, and diminished protein synthesis. The reduction in mTORC1 signalling was tightly coupled with increased expression and accumulation of REDD1 (regulated in DNA damage and development 1) and reduced phosphorylation of Akt and TSC2. The increase in REDD1 abundance was rapidly reversed upon iron repletion of cells but was also attenuated by inhibitors of gene transcription, protein phosphatase 2A (PP2A) and by REDD1 siRNA — strategies that also antagonised the loss in mTORC1 signalling associated with iron depletion. Our findings implicate REDD1 and PP2A as crucial regulators of mTORC1 activity in iron-depleted cells and indicate that their modulation may help mitigate atrophy of the intestinal mucosa that may occur in response to iron deficiency. PMID:26827808

  19. para-Sulphonato-calix[n]arenes as selective activators for the passage of molecules across the Caco-2 model intestinal membrane.

    Science.gov (United States)

    Roka, Eszter; Vecsernyes, Miklos; Bacskay, Ildiko; Félix, Caroline; Rhimi, Moez; Coleman, Anthony W; Perret, Florent

    2015-06-01

    The passage of Lucifer Yellow across the Caco-2 intestinal model membrane has been studied for the para-sulphonato-calix[n]arenes, the results show that para-sulphonato-calix[4]arene and para-sulphonato-calix[8]arene activate membrane passage when used simultaneously with a transport probe, Lucifer Yellow, whereas para-sulphonato-calix[6]arene has no effect. PMID:25958962

  20. Impact of anatase and rutile titanium dioxide nanoparticles on uptake carriers and efflux pumps in Caco-2 gut epithelial cells

    Science.gov (United States)

    Dorier, M.; Brun, E.; Veronesi, G.; Barreau, F.; Pernet-Gallay, K.; Desvergne, C.; Rabilloud, T.; Carapito, C.; Herlin-Boime, N.; Carrière, M.

    2015-04-01

    TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken together, these data suggest that TiO2-NPs may increase the functionality of gut epithelial cells, particularly their property to form a protective barrier against exogenous toxicants and to absorb nutrients.TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken

  1. Contributions of NanI Sialidase to Caco-2 Cell Adherence by Clostridium perfringens Type A and C Strains Causing Human Intestinal Disease

    OpenAIRE

    Li, Jihong; McClane, Bruce A.

    2014-01-01

    Previous studies showed that Clostridium perfringens type D animal disease strain CN3718 uses NanI sialidase for adhering to enterocyte-like Caco-2 cells. The current study analyzed whether NanI is similarly important when type A and C human intestinal disease strains attach to Caco-2 cells. A PCR survey determined that the nanI gene was absent from typical type A food poisoning (FP) strains carrying a chromosomal enterotoxin (CPE) gene or the genetically related type C Darmbrand (Db) strains...

  2. Contributions of NanI sialidase to Caco-2 cell adherence by Clostridium perfringens type A and C strains causing human intestinal disease.

    Science.gov (United States)

    Li, Jihong; McClane, Bruce A

    2014-11-01

    Previous studies showed that Clostridium perfringens type D animal disease strain CN3718 uses NanI sialidase for adhering to enterocyte-like Caco-2 cells. The current study analyzed whether NanI is similarly important when type A and C human intestinal disease strains attach to Caco-2 cells. A PCR survey determined that the nanI gene was absent from typical type A food poisoning (FP) strains carrying a chromosomal enterotoxin (CPE) gene or the genetically related type C Darmbrand (Db) strains. However, the nanI gene was present in type A strains from healthy humans, type A strains causing CPE-associated antibiotic-associated diarrhea (AAD) or sporadic diarrhea (SD), and type C Pig-Bel strains. Consistent with NanI sialidase being the major C. perfringens sialidase when produced, FP and Db strains had little supernatant sialidase activity compared to other type A or C human intestinal strains. All type A and C human intestinal strains bound to Caco-2 cells, but NanI-producing strains had higher attachment levels. When produced, NanI can contribute to host cell attachment of human intestinal disease strains, since a nanI null mutant constructed in type A SD strain F4969 had lower Caco-2 cell adhesion than wild-type F4969 or a complemented strain. Further supporting a role for NanI in host cell attachment, sialidase inhibitors reduced F4969 adhesion to Caco-2 cells. Collectively, these results suggest that NanI may contribute to the intestinal attachment and colonization needed for the chronic diarrhea of CPE-associated AAD and SD, but this sialidase appears to be dispensable for the acute pathogenesis of type A FP or type C enteritis necroticans. PMID:25135687

  3. The Staphylococcus aureus Alpha-Toxin Perturbs the Barrier Function in Caco-2 Epithelial Cell Monolayers by Altering Junctional Integrity

    OpenAIRE

    Kwak, Young-Keun; Vikström, Elena; Magnusson, Karl-Eric; Vécsey-Semjén, Beatrix; Colque-Navarro, Patricia; Möllby, Roland

    2012-01-01

    Increased microvascular permeability is a hallmark of sepsis and septic shock. Intestinal mucosal dysfunction may allow translocation of bacteria and their products, thereby promoting sepsis and inflammation. Although Staphylococcus aureus alpha-toxin significantly contributes to sepsis and perturbs the endothelial barrier function, little is known about possible effects of S. aureus alpha-toxin on human epithelial barrier functions. We hypothesize that S. aureus alpha-toxin in the blood can ...

  4. Indicaxanthin inhibits NADPH oxidase (NOX)-1 activation and NF-κB-dependent release of inflammatory mediators and prevents the increase of epithelial permeability in IL-1β-exposed Caco-2 cells.

    Science.gov (United States)

    Tesoriere, L; Attanzio, A; Allegra, M; Gentile, C; Livrea, M A

    2014-02-01

    Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1β, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1β brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signalling leading to the activation of NF-κB, with the over-expression of inflammatory enzymes and release of pro-inflammatory mediators. The co-incubation of the cells with Ind, at a nutritionally relevant concentration (5-25 μM), and IL-1β prevented the release of the pro-inflammatory cytokines IL-6 and IL-8, PGE2 and NO, the formation of ROS and the loss of thiols in a dose-dependent manner. The co-incubation of the cells with Ind and IL-1β also prevented the IL-1β-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-κB was prevented by Ind and the expression of COX-2 and inducible NO synthase was reduced. The uptake of Ind in Caco-2 cell monolayers appeared to be unaffected by the inflamed state of the cells. In conclusion, our findings suggest that the dietary pigment Ind may have the potential to modulate inflammatory processes at the intestinal level. PMID:23931157

  5. 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...

  6. Effect of edible oils on quercetin, kaempferol and galangin transport and conjugation in the intestinal Caco-2/HT29-MTX co-culture model

    OpenAIRE

    Jailani, F; Williamson, G

    2014-01-01

    Solubility and matrix play an important role in the gut lumen in delivering bioactive compounds to the absorptive surface of enterocytes. The purpose of this study was to determine the effect of certain commonly consumed lipids, soybean, olive and corn oil, on the transport and conjugation of flavonols (myricetin, quercetin, kaempferol and galangin) using the conjugation-competent co-cultured Caco-2/HT29-MTX intestinal cell monolayer model. To enable identification and quantification of conju...

  7. 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

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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

  8. Targeted drug delivery systems 6: Intracellular bioreductive activation, uptake and transport of an anticancer drug delivery system across intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Gharat, L; Taneja, R; Weerapreeyakul, N; Rege, B; Polli, J; Chikhale, P J

    2001-05-21

    We demonstrate transport across, intracellular accumulation and bioreductive activation of a conformationally constrained, anticancer drug delivery system (the CH(3)-TDDS) using Caco-2 cell monolayers (CCMs) as an in vitro model of the human intestinal mucosa. Reverse-phase High Performance Liquid Chromatography (HPLC) coupled with UV detection was used to detect CH(3)-TDDS, the bioreduction product (lactone) and the released drug (melphalan methyl ester; MME). Upon incubation of the CH(3)-TDDS with the apical (AP) surface of 21-day-old CCM, we observed rapid decrease in the AP concentration of the CH(3)-TDDS (60%/hr) as a result of cellular uptake. Rapid intracellular accumulation of the CH(3)-TDDS was followed by bioreductive activation to deplete the cellular levels of CH(3)-TDDS. The drug part (MME) and lactone, as well as CH(3)-TDDS, were detected in the basolateral (BL) chamber. Intracellular Caco-2 levels of TDDS and lactone were also detectable. Bioreductive activation of the CH(3)-TDDS was additionally confirmed by formation of lactone after incubation of the CH(3)-TDDS in the presence of freshly prepared Caco-2 cell homogenates. During transport studies of melphalan or MME alone (as control), the intact drug was not detected in the intracellular compartment or in the BL chamber. These observations demonstrate that CH(3)-TDDS has potential for improving intestinal delivery of MME. TDDS could be useful in facilitating oral absorption of MME as well as the oral delivery of other agents. PMID:11337161

  9. Tuning the inflammatory response to silver nanoparticles via quercetin in Caco-2 (co-)cultures as model of the human intestinal mucosa.

    Science.gov (United States)

    Martirosyan, Alina; Grintzalis, Konstantinos; Polet, Madeleine; Laloux, Laurie; Schneider, Yves-Jacques

    2016-06-24

    Interaction of nanoparticles with food matrix components may cause unpredictable health complications. Using an improved Caco-2 cell-based in vitro (co-)culture model the potential of quercetin as one of the major food flavonoids to alter the effect of silver nanoparticles (Ag-NPs) <20 nm in the human intestinal mucosa at real life concentrations was investigated. Ag-NPs (15-90 μg/ml) decreased cell viability and reduced thiol groups, induced oxidative/nitrosative stress and lipid peroxidation and led to activity changes of various antioxidant enzymes after 3h exposure. The contribution of Ag(+) ions within the concentrations released from nanoparticles was shown to be less important, compared to Ag-NPs. While leading to inflammatory response in the intestines, Ag-NPs, paradoxically, also showed a potential anti-infammatory effect manifested in down-regulated IL-8 levels. Quercetin, co-administered with Ag-NPs, led to a reduction of cytotoxicity, oxidative stress, and recovered metabolic activity of Caco-2 cells, suggesting the protective effects of this flavonoid against the harmful effect of Ag-NPs. Quercetin not only alleviated the effect of Ag-NPs on the gastrointestinal cells, but also demonstrated a potential to serve as a tool for reversible modulation of intestinal permeability. PMID:27113704

  10. The effect of phytic acid on tight junctions in the human intestinal Caco-2 cell line and its mechanism.

    Science.gov (United States)

    Fu, Qingxue; Wang, Huizhen; Xia, Mengxin; Deng, Bing; Shen, Hongyi; Ji, Guang; Li, Guowen; Xie, Yan

    2015-12-01

    This study investigated the effect of phytic acid (IP6), a potential absorption enhancer of flavonoid components, on tight junction (TJ) integrity in Caco-2 cell monolayers and its possible mechanisms. Transepithelial electrical resistance (TEER) across the monolayers decreased rapidly, and the flux of fluorescein sodium (a paracellular marker) increased after treating with IP6 in a concentration-dependent manner. Confocal microscopy results showed that IP6 produced a concentration-dependent attenuation in the distribution of occludin, ZO-1, and claudin-1. Immunoblot analysis revealed that IP6 could down-regulate the expression level of these TJ proteins, which resulted in the opening of TJ. Additionally, the divalent cations Ca(2+) and Mg(2+) influenced the IP6-induced distribution of occludin, ZO-1, and claudin-1 in different directions, which enhanced barrier function. In conclusion, IP6 can decrease the integrity of Caco-2 cell monolayers by modulating the TJ proteins' localization and down-regulating the expression levels of TJ proteins including claudin-1, occludin, and ZO-1; the reduction effects of divalent cations such as Ca(2+) and Mg(2+) on the regulation of TJ induced by IP6 should be addressed. The present work will offer some useful guidance for the application of IP6 in drug delivery area. PMID:26385515

  11. Intestinal Epithelial Cells In Vitro

    OpenAIRE

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

    2009-01-01

    Recent advances in the biology of stem cells has resulted in significant interest in the development of normal epithelial cell lines from the intestinal mucosa, both to exploit the therapeutic potential of stem cells in tissue regeneration and to develop treatment models of degenerative disorders of the digestive tract. However, the difficulty of propagating cell lines of normal intestinal epithelium has impeded research into the molecular mechanisms underlying differentiation of stem/progeni...

  12. 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...

  13. Contribution of Listeria monocytogenes RecA to acid and bile survival and invasion of human intestinal Caco-2 cells

    NARCIS (Netherlands)

    Veen, van der S.; Abee, T.

    2011-01-01

    The food-borne pathogen Listeria monocytogenes is able to colonize the human gastro-intestinal tract and subsequently cross the intestinal barrier. Thus, for L. monocytogenes to become virulent, it must survive the low pH of the stomach, high bile concentrations in the small intestine, and invade th

  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

    2016-04-20

    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. PMID

  15. 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

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

    Science.gov (United States)

    Drummond, Coyne G.

    2015-01-01

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

  17. 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 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 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. 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.

  18. Functional Comparison of Human Colonic Carcinoma Cell Lines and Primary Small Intestinal Epithelial Cells for Investigations of Intestinal Drug Permeability and First-Pass Metabolism.

    Science.gov (United States)

    Yamaura, Yoshiyuki; Chapron, Brian D; Wang, Zhican; Himmelfarb, Jonathan; Thummel, Kenneth E

    2016-03-01

    To further the development of a model for simultaneously assessing intestinal absorption and first-pass metabolism in vitro, Caco-2, LS180, T84, and fetal human small intestinal epithelial cells (fSIECs) were cultured on permeable inserts, and the integrity of cell monolayers, CYP3A4 activity, and the inducibility of enzymes and transporters involved in intestinal drug disposition were measured. Caco-2, T84, and fSIECs all formed tight junctions, as assessed by immunofluorescence microscopy for zonula occludens-1, which was well organized into circumscribing strands in T84, Caco-2, and fSIECs but was diffuse in LS180 cells. The transepithelial electrical resistance value for LS180 monolayers was lower than that for Caco-2, T84, and fSIECs. In addition, the apical-to-basolateral permeability of the paracellular marker Lucifer yellow across LS180 monolayers was greater than in fSIECs, T84, and Caco-2 monolayers. The transcellular marker propranolol exhibited similar permeability across all cells. With regard to metabolic capacity, T84 and LS180 cells showed comparable basal midazolam hydroxylation activity and was inducible by rifampin and 1α,25(OH)2D3 in LS180 cells, but only marginally so in T84 cells. The basal CYP3A4 activity of fSIECs and Caco-2 cells was much lower and not inducible. Interestingly, some of the drug transporters expressed in LS180 and Caco-2 cells were induced by either 1α,25(OH)2D3 or rifampin or both, but effects were limited in the other two cell lines. These results suggest that none of the cell lines tested fully replicated the drug disposition properties of the small intestine and that the search for an ideal screening tool must continue. PMID:26700954

  19. Aflatoxin M1 cytotoxicity against human intestinal Caco-2 cells is enhanced in the presence of other mycotoxins.

    Science.gov (United States)

    Gao, Y N; Wang, J Q; Li, S L; Zhang, Y D; Zheng, N

    2016-10-01

    Aflatoxin M1 (AFM1), a class 2B human carcinogen, is the only mycotoxin with established maximum residue limits (MRLs) in milk. Toxicological data for other mycotoxins in baby food, containing cereals and milk, either in isolation or in combination with AFM1, are sparse. The aim of this study was to investigate the cytotoxicity of AFM1, ochratoxin A (OTA), zearalenone (ZEA), and α-zearalenol (α-ZOL), individually and in combinations, in human Caco-2 cells. The tetrazolium salt (MTT) assay demonstrated that (i) OTA and AFM1 had similar cytotoxicity, which was higher than that of ZEA and α-ZOL, after a 72 h exposure; and (ii) the quaternary combination had the highest cytotoxicity, followed by tertiary and binary combinations and individual mycotoxins. Isobologram analysis indicated that the presence of OTA, ZEA, and/or α-ZOL with AFM1 led to additive and synergistic cytotoxicity in most combinations. The cytotoxicity of OTA was similar to that of AFM1, suggesting that OTA in food poses a health risk to consumers. Furthermore, AFM1 cytotoxicity increased dramatically in the presence of OTA, ZEA, and/or α-ZOL (p mycotoxins in baby food which contains milk and cereals. PMID:27470613

  20. 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. PMID:27131754

  1. 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. PMID:27220052

  2. Butyrate Produced by Commensal Bacteria Potentiates Phorbol Esters Induced AP-1 Response in Human Intestinal Epithelial Cells

    OpenAIRE

    Nepelska, Malgorzata; Cultrone, Antonietta; Béguet-Crespel, Fabienne; Le Roux, Karine; Doré, Joël; Arulampalam, Vermulugesan; Blottière, Hervé M.

    2012-01-01

    The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect...

  3. Hepatocyte nuclear factor 1 coordinates multiple processes in a model of intestinal epithelial cell function.

    Science.gov (United States)

    Yang, Rui; Kerschner, Jenny L; Harris, Ann

    2016-04-01

    Mutations in hepatocyte nuclear factor 1 transcription factors (HNF1α/β) are associated with diabetes. These factors are well studied in the liver, pancreas and kidney, where they direct tissue-specific gene regulation. However, they also have an important role in the biology of many other tissues, including the intestine. We investigated the transcriptional network governed by HNF1 in an intestinal epithelial cell line (Caco2). We used chromatin immunoprecipitation followed by direct sequencing (ChIP-seq) to identify HNF1 binding sites genome-wide. Direct targets of HNF1 were validated using conventional ChIP assays and confirmed by siRNA-mediated depletion of HNF1, followed by RT-qPCR. Gene ontology process enrichment analysis of the HNF1 targets identified multiple processes with a role in intestinal epithelial cell function, including properties of the cell membrane, cellular response to hormones, and regulation of biosynthetic processes. Approximately 50% of HNF1 binding sites were also occupied by other members of the intestinal transcriptional network, including hepatocyte nuclear factor 4A (HNF4A), caudal type homeobox 2 (CDX2), and forkhead box A2 (FOXA2). Depletion of HNF1 in Caco2 cells increases FOXA2 abundance and decreases levels of CDX2, illustrating the coordinated activities of the network. These data suggest that HNF1 plays an important role in regulating intestinal epithelial cell function, both directly and through interactions with other intestinal transcription factors. PMID:26855178

  4. The Intestine Plays a Substantial Role in Human Vitamin B6 Metabolism: A Caco-2 Cell Model

    OpenAIRE

    Albersen, Monique; Bosma, Marjolein; Knoers, Nine V. V. A. M.; de Ruiter, Berna H. B.; Diekman, Eugène F.; de Ruijter, Jessica; Visser, Wouter F.; de Koning, Tom J.; Verhoeven-Duif, Nanda M.

    2013-01-01

    Background Vitamin B6 is present in various forms (vitamers) in the diet that need to be metabolized to pyridoxal phosphate (PLP), the active cofactor form of vitamin B6. In literature, the liver has been reported to be the major site for this conversion, whereas the exact role of the intestine remains to be elucidated. Objective To gain insight into the role of the intestine in human vitamin B6 metabolism. Materials and Methods Expression of the enzymes pyridoxal kinase (PK), pyridox(am)ine ...

  5. The Intestine Plays a Substantial Role in Human Vitamin B6 Metabolism : A Caco-2 Cell Model

    NARCIS (Netherlands)

    Albersen, Monique; Bosma, Marjolein; Knoers, Nine V. V. A. M.; de Ruiter, Berna H. B.; Diekman, Eugene F.; de Ruijter, Jessica; Visser, Wouter F.; de Koning, Tom J.; Verhoeven-Duif, Nanda M.

    2013-01-01

    Background: Vitamin B6 is present in various forms (vitamers) in the diet that need to be metabolized to pyridoxal phosphate (PLP), the active cofactor form of vitamin B6. In literature, the liver has been reported to be the major site for this conversion, whereas the exact role of the intestine rem

  6. CriticalSorb promotes permeation of flux markers across isolated rat intestinal mucosae and Caco-2 monolayers

    OpenAIRE

    Brayden, David James; Bzik, V. A.; Lewis, A L; Illum, L.

    2012-01-01

    Purpose CriticalSorb™ is a novel absorption enhancer based on Solutol® HS15, one that has been found to enhance the nasal transport. It is in clinical trials for nasal delivery of human growth hormone. The hypothesis was that permeating enhancement effects of the Solutol®HS15 component would translate to the intestine. Methods Rat colonic mucosae were mounted in Ussing chambers and Papp values of [14C]-mannitol, [14C]-antipyrine, FITC-dextran 4000 (FD-4), and TEER values were calcul...

  7. Apoptosis induced by oxidized lipids is associated with up-regulation of p66Shc in intestinal Caco-2 cells: protective effects of phenolic compounds.

    Science.gov (United States)

    Giovannini, Claudio; Scazzocchio, Beatrice; Matarrese, Paola; Varì, Rosaria; D'Archivio, Massimo; Di Benedetto, Roberta; Casciani, Stefania; Dessì, Maria Rita; Straface, Elisabetta; Malorni, Walter; Masella, Roberta

    2008-02-01

    In this study, we investigated the alterations of the redox balance induced by the lipid fraction of oxLDL in Caco-2 intestinal cells, and the effects of tyrosol and protocatechuic acid, two dietary phenolic compounds. We found that oxidized lipids extracted from oxLDL (LipE) induced oxidative stress by determining, 6 h after treatment, ROS overproduction (about a 100% and a 43% increase of O*2 and H2O2 production, respectively, P<.05: LipE vs. control) and, 12 h after treatment, GSH depletion (about a 26% decrease, P<.05: LipE vs. control), and by impairing the activities of superoxide dismutase, catalase and glutathione peroxidase. In response to the induced oxidative stress, we observed significant overexpression of glutathione peroxidase (6 h after treatment: P<.05), glutathione reductase and gamma-glutamylcysteine synthetase (12 h after treatment: P<.05). Notably, when GSH depletion occurred, p66Shc protein expression increased by about 300% with respect to control (P<.001; LipE vs. control). These effects were fully counteracted by dietary phenolics which inhibited ROS overproduction and GSH consumption, rendered the reactive transcription of glutathione-associated enzymes unnecessary and blocked the intracellular signals leading to the overexpression and rearrangement of p66Shc signalling molecule. Altogether, these results suggest that the impairment of the antioxidant system hijacks intestinal cells towards an apoptotic-prone phenotype via the activation of p66Shc molecule. They also propose a reappraisal of dietary polyphenols as intestinal protecting agents, indicating the antiapoptotic effect as a further mechanism of action of these antioxidant compounds. PMID:17588737

  8. Dysregulated expression of arginine metabolic enzymes in human intestinal tissues of necrotizing enterocolitis and response of CaCO2 cells to bacterial components.

    Science.gov (United States)

    Leung, Kam Tong; Chan, Kathy Yuen Yee; Ma, Terence Ping Yuen; Yu, Jasmine Wai Sum; Tong, Joanna Hung Man; Tam, Yuk Him; Cheung, Hon Ming; To, Ka Fai; Lam, Hugh Simon; Lee, Kim Hung; Li, Karen; Ng, Pak Cheung

    2016-03-01

    The small intestine is the exclusive site of arginine synthesis in neonates. Low levels of circulating arginine have been associated with the occurrence of necrotizing enterocolitis (NEC) but the mechanism of arginine dysregulation has not been fully elucidated. We aimed to investigate (i) expressional changes of arginine synthesizing and catabolic enzymes in human intestinal tissues of NEC, spontaneous intestinal perforation (SIP) and noninflammatory surgical conditions (Surg-CTL) and to investigate the (ii) mechanisms of arginine dysregulation and enterocyte proliferation upon stimulation by bacterial components, arginine depletion, ARG1 overexpression and nitric oxide (NO) supplementation. Our results showed that expressions of arginine synthesizing enzymes ALDH18A1, ASL, ASS1, CPS1, GLS, OAT and PRODH were significantly decreased in NEC compared with Surg-CTL or SIP tissues. Catabolic enzyme ARG1 was increased (>100-fold) in NEC tissues and histologically demonstrated to be expressed by infiltrating neutrophils. No change in arginine metabolic enzymes was observed between SIP and Surg-CTL tissues. In CaCO2 cells, arginine metabolic enzymes were differentially dysregulated by lipopolysaccharide or lipoteichoic acid. Depletion of arginine reduced cell proliferation and this phenomenon could be partially rescued by NO. Overexpression of ARG1 also reduced enterocyte proliferation. We provided the first expressional profile of arginine metabolic enzymes at the tissue level of NEC. Our findings suggested that arginine homeostasis was severely disturbed and could be triggered by inflammatory responses of enterocytes and infiltrating neutrophils as well as bacterial components. Such reactions could reduce arginine and NO, resulting in mucosal damage. The benefit of arginine supplementation for NEC prophylaxis merits further clinical evaluation. PMID:26895666

  9. 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

    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...

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

    Science.gov (United States)

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

    2016-01-14

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

  11. 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. PMID:27067107

  12. Intestinal epithelial cells in inflammatory bowel diseases

    Institute of Scientific and Technical Information of China (English)

    Giulia; Roda; Alessandro; Sartini; Elisabetta; Zambon; Andrea; Calafiore; Margherita; Marocchi; Alessandra; Caponi; Andrea; Belluzzi; Enrico; Roda

    2010-01-01

    The pathogenesis of inflammatory bowel diseases (IBDs) seems to involve a primary defect in one or more of the elements responsible for the maintenance of intestinal homeostasis and oral tolerance. The most important element is represented by the intestinal barrier, a complex system formed mostly by intestinal epithelial cells (IECs). IECs have an active role in producing mucus and regulating its composition; they provide a physical barrier capable of controlling antigen traff ic through the intestinal muco...

  13. 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

    2016-01-01

    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 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. PMID:27399765

  14. Wound healing of intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Shiho Konno

    2011-01-01

    Full Text Available The intestinal epithelial cells (IECs form a selective permeability barrier separating luminal content from underlying tissues. Upon injury, the intestinal epithelium undergoes a wound healing process. Intestinal wound healing is dependent on the balance of three cellular events; restitution, proliferation, and differentiation of epithelial cells adjacent to the wounded area. Previous studies have shown that various regulatory peptides, including growth factors and cytokines, modulate intestinal epithelial wound healing. Recent studies have revealed that novel factors, which include toll-like receptors (TLRs, regulatory peptides, particular dietary factors, and some gastroprotective agents, also modulate intestinal epithelial wound repair. Among these factors, the activation of TLRs by commensal bacteria is suggested to play an essential role in the maintenance of gut homeostasis. Recent studies suggest that mutations and dysregulation of TLRs could be major contributing factors in the predisposition and perpetuation of inflammatory bowel disease. Additionally, studies have shown that specific signaling pathways are involved in IEC wound repair. In this review, we summarize the function of IECs, the process of intestinal epithelial wound healing, and the functions and mechanisms of the various factors that contribute to gut homeostasis and intestinal epithelial wound healing.

  15. De-phosphorylation of TRα-1 by p44/42 MAPK inhibition enhances T3-mediated GLUT5 gene expression in the intestinal cell line Caco-2 cells

    International Nuclear Information System (INIS)

    Thyroid hormone and p44/42 MAPK inactivation are important in intestinal differentiation. We demonstrated not only that treatment with p44/42 MAPK inhibitor U0126 in intestinal cell line Caco-2 cells reduced the phosphorylation of serine and threonine residues of TRα-1, but also that T3 and U0126 synergistically induced GLUT5 gene expression. EMSA demonstrated that the binding activity of TRα-1-RXR heterodimer on GLUT5-TRE in nuclear proteins of Caco-2 cells was synergistically enhanced by co-incubation in vitro with T3 and CIAP, which strongly de-phosphorylates proteins. ChIP and transfection assays revealed that co-treatment of T3 and U0126 induces TRα-1-RXR binding to GLUT5-TRE on the human GLUT5 enhancer region, and recruitment of the transcriptional complex in cells. These results suggest that inactivation of p44/42 MAPK enhances T3-induced GLUT5 gene expression in Caco-2 cells through increasing TRα-1 transactivity and binding activity to the GLUT5-TRE, probably due to de-phosphorylation of TRα-1

  16. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    International Nuclear Information System (INIS)

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [3H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [3H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-κB, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  17. A novel anti-inflammatory role of GPR120 in intestinal epithelial cells.

    Science.gov (United States)

    Anbazhagan, Arivarasu N; Priyamvada, Shubha; Gujral, Tarunmeet; Bhattacharyya, Sumit; Alrefai, Waddah A; Dudeja, Pradeep K; Borthakur, Alip

    2016-04-01

    GPR120 (free fatty acid receptor-4) is a G protein-coupled receptor for medium- and long-chain unsaturated fatty acids, including ω-3 fatty acids. Recent studies have shown GPR120 to play cardinal roles in metabolic disorders via modulation of gut hormone secretion and insulin sensitivity and to exert anti-inflammatory effects in macrophages and adipose tissues. However, information on anti-inflammatory role of GPR120 at the level of intestinal epithelium is very limited. Current studies demonstrated differential levels of GPR120 mRNA and protein along the length of the human, mouse, and rat intestine and delineated distinct anti-inflammatory responses following GPR120 activation in model human intestinal epithelial Caco-2 cells, but not in model mouse intestinal epithelial endocrine cell line STC-1. In Caco-2 cells, GPR120 was internalized, bound to β-arrestin-2, and attenuated NF-κB activation in response to 30-min exposure to the agonists GW9508, TUG-891, or docosahexaenoic acid. These effects were abrogated in response to small interfering RNA silencing of β-arrestin-2. Treatment of STC-1 cells with these agonists did not induce receptor internalization and had no effects on NF-κB activation, although treatment with the agonists GW9508 or TUG-891 for 6 h augmented the synthesis and secretion of the gut hormone glucagon-like peptide-1 in this cell line. Our studies for the first time demonstrated a GPR120-mediated novel anti-inflammatory pathway in specific intestinal epithelial cell types that could be of therapeutic relevance to intestinal inflammatory disorders. PMID:26791484

  18. Microecology, intestinal epithelial barrier and necrotizing enterocolitis

    OpenAIRE

    Sharma, Renu; Tepas, Joseph J.

    2009-01-01

    Soon after birth, the neonatal intestine is confronted with a massive antigenic challenge of microbial colonization. Microbial signals are required for maturation of several physiological, anatomical, and biochemical functions of intestinal epithelial barrier (IEB) after birth. Commensal bacteria regulate intestinal innate and adaptive immunity and provide stimuli for ongoing repair and restitution of IEB. Colonization by pathogenic bacteria and/or dysmature response to microbial stimuli can ...

  19. Human influenza A viruses are proteolytically activated and do not induce apoptosis in CACO-2 cells

    International Nuclear Information System (INIS)

    Replication of human influenza A/H3N2 and A/H1N1 viruses was studied in human CACO-2 cells, a continuous line of intestinal epithelial differentiated cells. Hemagglutinin (HA) was cleaved in these cells by an endogenous protease. Thus, infectious virus was produced that underwent multiple cycle replication and plaque formation in the absence of trypsin added to the media. Cleavage of de novo-synthesized HA occurred at a late stage of the exocytic pathway as indicated by pulse-chase labeling and by experiments employing endoglycosidase H and brefeldin A treatment. However, surface-labeling experiments employing biotinylation suggested that there is no cleavage at the plasma membrane. Unlike HA of serotypes H5 and H7 cleaved at multibasic cleavage sites by furin, the HAs with monobasic cleavage sites analyzed here were not cleaved in CACO-2 cells in the presence of aprotinin, a natural inhibitor of trypsinlike proteases. Growing CACO-2 cells were able to cleave HA of incoming virus, although influenza virus activating protease was not detected in culture medium. These observations indicate that the activating enzyme of CACO-2 cells is a trypsinlike protease functioning in the trans-Golgi network and presumably endosomes. In support of this concept immune staining with antibodies specific to human and bovine trypsin revealed the presence of a trypsinlike protease in CACO-2 cells. Unlike MDCK and CV-1 cells undergoing rapid apoptosis after influenza virus infection, CACO-2 cells showed no apoptosis but displayed cytopathic effects with necrotic signs significantly later after infection. It follows from these data that, depending on the cell type, influenza virus may kill cells either by apoptosis or by necrosis

  20. Dissecting stromal-epithelial interactions in a 3D in vitro cellularized intestinal model for permeability studies.

    Science.gov (United States)

    Pereira, Carla; Araújo, Francisca; Barrias, Cristina C; Granja, Pedro L; Sarmento, Bruno

    2015-07-01

    Absorption evaluation plays an increasingly important role at the early stage of drug discovery due to its potential to scan the ADME (absorption, distribution, metabolism and excretion) properties of new drug candidates. Therefore, a new three-dimensional (3D) in vitro model replicating the intestinal functioning is herein proposed aiming to dissect the stromal-epithelial interactions and evaluate the permeation of a model drug, insulin. Inspired on the intestinal mucosal architecture, the present model comprises intestinal myofibroblasts (CCD18-Co cells) embedded in Matrigel, onto which epithelial enterocytes (Caco-2 cells) and mucus-producing cells (HT29-MTX cells) were seeded. CCD18-Co myofibroblasts showed to have a central role in the remodeling of the surrounding matrix confirmed by the production of fibronectin. Subsequently, this matrix revealed to be essential to the maintenance of the model architecture by supporting the overlying epithelial cells. In terms of functionality, this model allowed the efficient prediction of insulin permeability in which the presence of mucus, the less tight character between Caco-2 and HT29-MTX epithelial cells and the 3D assembly were critical factors. Concluding, this model constitutes a robust tool in the drug development field with potential to bridge the traditional 2D cell culture models and in vivo animal models. PMID:25934277

  1. Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

    Science.gov (United States)

    Finamore, Alberto; Roselli, Marianna; Imbinto, Ambra; Seeboth, Julie; Oswald, Isabelle P.; Mengheri, Elena

    2014-01-01

    Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans

  2. Lactobacillus amylovorus inhibits the TLR4 inflammatory signaling triggered by enterotoxigenic Escherichia coli via modulation of the negative regulators and involvement of TLR2 in intestinal Caco-2 cells and pig explants.

    Science.gov (United States)

    Finamore, Alberto; Roselli, Marianna; Imbinto, Ambra; Seeboth, Julie; Oswald, Isabelle P; Mengheri, Elena

    2014-01-01

    Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans

  3. 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. PMID:26791917

  4. Characterization of the intestinal absorption of seven flavonoids from the flowers of Trollius chinensis using the Caco-2 cell monolayer model.

    Directory of Open Access Journals (Sweden)

    Lijia Liu

    Full Text Available The human Caco-2 cell monolayer model was used to investigate the absorption property, mechanism, and structure-property relationship of seven representative flavonoids, namely, orientin, vitexin, 2"-O-β-L-galactopyranosylorientin, 2"-O-β-L-galactopyranosylvitexin, isoswertisin, isoswertiajaponin, and 2"-O-(2"'-methylbutanoylisoswertisin from the flowers of Trollius chinensis. The results showed that these flavonoids were hardly transported through the Caco-2 cell monolayer. The compounds with 7-OCH3 including isoswertisin, isoswertiajaponin and 2"-O-(2"'-methylbutanoylisoswertisin were absorbed in a passive diffusion manner, and their absorbability was increased in the same order as their polarity. The absorption of the remaining compounds with 7-OH including orientin, vitexin, 2"-O-β-L-galactopyranosylorientin, and 2"-O-β-L-galactopyranosylvitexin involved transporter mediated efflux in addition to passive diffusion. Among the four compounds with 7-OH, those with a free hydroxyl group at C-2" such as orientin and vitexin were the substrates of P-glycoprotein (P-gp and that with a free hydroxyl group at C-2' such as 2"-O-β-L-galactopyranosylorientin was the substrate of multidrug resistance protein 2 (MRP2. The results of this study also implied that the absorbability of the flavonoids should be taken into account when estimating the effective components of T. chinensis.

  5. In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.

    Science.gov (United States)

    Fiorentino, Maria; Lammers, Karen M; Levine, Myron M; Sztein, Marcelo B; Fasano, Alessio

    2013-01-01

    Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates. PMID:23408152

  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. The effects of hydrothermal processing and germination on Fe speciation and Fe bioaccessibility to human intestinal Caco-2 cells in Tartary buckwheat.

    Science.gov (United States)

    Pongrac, Paula; Scheers, Nathalie; Sandberg, Ann-Sofie; Potisek, Mateja; Arčon, Iztok; Kreft, Ivan; Kump, Peter; Vogel-Mikuš, Katarina

    2016-05-15

    Tartary buckwheat is a gluten-free crop with great potential as a wheat substitute. Iron (Fe) is an important mineral element in staple foods which is required in sufficient bioaccessible quantities. The aim of the study was to investigate how processing of grains into groats (hydrothermal processing to remove the husk) and sprouts (7-day-old seedlings) affected Fe speciation (Fe(2+) or Fe(3+)), Fe ligand composition and Fe bioaccessibility to human Caco-2 cells. Groats contained the least Fe (23.8 ± 1.65 mg kg(-1)) and the lowest amounts of Fe(2+) (8%). Grains and sprouts had comparable Fe concentrations (78.2 ± 2.65 and 68.9 ± 2.73 mg kg(-1)) and similar proportions of Fe(2+) (15% and 18%). The main ligands for Fe in Tartary buckwheat material were phytate and citrate. Phytate was less abundant in sprouts, which did not correlate with greater Fe bioaccessibility. Iron bioaccessibility was 4.5-fold greater for grains than groats, suggesting that Fe is more bioaccessible in the husk than in the rest of the grain. PMID:26776035

  8. Lactobacillus reuteri glyceraldehyde-3-phosphate dehydrogenase functions in adhesion to intestinal epithelial cells.

    Science.gov (United States)

    Zhang, Wen-Ming; Wang, Hai-Feng; Gao, Kan; Wang, Cong; Liu, Li; Liu, Jian-Xin

    2015-05-01

    This study was aimed to identify key surface proteins mediating the adhesion of lactobacilli to intestinal epithelial cells. By using Caco-2 and IPEC-J2 cells labeled with sulfo-NHS-biotin in the western blotting, a protein band of an approximately 37 kDa was detected on the surface layer of Lactobacillus reuteri strains ZJ616, ZJ617, ZJ621, and ZJ623 and Lactobacillus rhamnosus GG. Mass spectrometry analysis using the adhesion-related protein from L. reuteri ZJ617 showed that it was 100% homologous to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. reuteri JCM 1112 (GenBank: YP_001841377). The ability of L. reuteri ZJ617 to adhere to epithelial cells decreased significantly by treatment with LiCl or by blocking with an anti-GAPDH antibody, in comparison with the untreated strain (p < 0.05). Immunoelectron microscopic and immunofluorescence analyses confirmed that GAPDH is located on the surface layer of L. reuteri ZJ617. The results indicated that the GAPDH protein of L. reuteri ZJ617 acts as an adhesion component that plays an important role in binding to the intestinal epithelial cells. PMID:25867279

  9. Inhibitory effect of carnosine on interleukin-8 production in intestinal epithelial cells through translational regulation.

    Science.gov (United States)

    Son, Dong Ok; Satsu, Hideo; Kiso, Yoshinobu; Totsuka, Mamoru; Shimizu, Makoto

    2008-05-01

    The enhanced intestinal production of pro-inflammatory cytokines leads to inflammation and carcinogenesis, and therefore its down-regulation by nutrients could represent a promising therapeutic approach. We found for the first time that the secretion of interleukin-8 (IL-8) in intestinal epithelial cells stimulated by hydrogen peroxide or TNF-alpha was suppressed in the presence of carnosine (beta-Ala-His), a dietary dipeptide. Interestingly, carnosine had no influence on the stimulus-induced IL-8 mRNA expression, although the intracellular production and secretion of IL-8 were significantly inhibited by carnosine. The inhibitory effect of carnosine on the IL-8 secretion differed from that of other histidine-containing dipeptides like Gly-His, Ala-His, and anserine (beta-Ala-1-methyl-His), which inhibited both the hydrogen peroxide-induced secretion and mRNA expression of IL-8. These observations indicate that carnosine inhibited IL-8 secretion along a unique pathway, in which IL-8 production was suppressed at a post-transcriptional level, for instance, translation. The hypothesis that carnosine inhibited the translation of IL-8 mRNA is supported by the finding that the phosphorylation of eIF4E, an initiation factor, in stimulated Caco-2 cells was inhibited by carnosine. These results suggest that carnosine is a novel type of anti-inflammatory agent that down-regulates the inflammatory response in intestinal epithelial cells by a unique mechanism. PMID:18397832

  10. Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

    Science.gov (United States)

    Cundiff, Judy K; McConnell, Elizabeth J; Lohe, Kimberly J; Maria, Sarah D; McMahon, Robert J; Zhang, Qiang

    2016-01-01

    Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development. PMID:26586228

  11. Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro.

    Science.gov (United States)

    Jensen, Hanne; Roos, Stefan; Jonsson, Hans; Rud, Ida; Grimmer, Stine; van Pijkeren, Jan-Peter; Britton, Robert A; Axelsson, Lars

    2014-04-01

    Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA). PMID:24473252

  12. Constitutive activation of the MEK/ERK pathway inhibits intestinal epithelial cell differentiation.

    Science.gov (United States)

    Lemieux, Etienne; Boucher, Marie-Josée; Mongrain, Sébastien; Boudreau, François; Asselin, Claude; Rivard, Nathalie

    2011-10-01

    The Ras/Raf/MEK/ERK cascade regulates intestinal epithelial cell proliferation. Indeed, while barely detectable in differentiated cells of the villi, ERK1/2-activated forms are detected in the nucleus of undifferentiated human intestinal crypt cells. In addition, we and others have reported that ERKs are selectively inactivated during enterocyte differentiation. However, whether inactivation of the ERK pathway is necessary for inhibition of both proliferation and induction of differentiation of intestinal epithelial cells is unknown. Human Caco-2/15 cells, undifferentiated crypt IEC-6 cells, and differentiating Cdx3-expressing IEC-6 cells were infected with retroviruses encoding either a hemagglutinin (HA)-tagged MEK1 wild type (wtMEK) or a constitutively active S218D/S222D MEK1 mutant (caMEK). Protein and gene expression was assessed by Western blotting, semiquantitative RT-PCR, and real-time PCR. Morphology was analyzed by transmission electron microscopy. We found that 1) IEC-6/Cdx3 cells formed multicellular layers after confluence and differentiated after 30 days in culture, as assessed by increased polarization, microvilli formation, expression of differentiation markers, and ERK1/2 inhibition; 2) while activated MEK prevented neither the inhibition of ERK1/2 activities nor the differentiation process in postconfluent Caco-2/15 cells, caMEK expression prevented ERK inhibition in postconfluent IEC-6/Cdx3 cells, thus leading to maintenance of elevated ERK1/2 activities; 3) caMEK-expressing IEC-6/Cdx3 cells exhibited altered multicellular structure organization, poorly defined tight junctions, reduced number of microvilli on the apical surface, and decreased expression of the hepatocyte nuclear factor 1α transcription factor and differentiation markers, namely apolipoprotein A-4, fatty acid-binding protein, calbindin-3, mucin 2, alkaline phosphatase, and sucrase-isomaltase; and 4) increased Cdx3 phosphorylation on serine-60 (S60) in IEC-6/Cdx3 cells expressing

  13. 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.

  14. 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.

    Science.gov (United States)

    Nzakizwanayo, Jonathan; Kumar, Sandeep; Ogilvie, Lesley A; Patel, Bhavik A; Dedi, Cinzia; Macfarlane, Wendy M; Jones, Brian V

    2015-01-01

    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. PMID:25790373

  15. Apoptosis of human intestinal epithelial cells after bacterial invasion.

    OpenAIRE

    Kim, J. M.; Eckmann, L; Savidge, T. C.; Lowe, D C; Witthöft, T; Kagnoff, M F

    1998-01-01

    Epithelial cells that line the human intestinal mucosa are the initial site of host invasion by bacterial pathogens. The studies herein define apoptosis as a new category of intestinal epithelial cell response to bacterial infection. Human colon epithelial cells are shown to undergo apoptosis following infection with invasive enteric pathogens, such as Salmonella or enteroinvasive Escherichia coli. In contrast to the rapid onset of apoptosis seen after bacterial infection of mouse monocyte-ma...

  16. Regulation of intracellular Zn homeostasis in two intestinal epithelial cell models at various maturation time points.

    Science.gov (United States)

    Gefeller, Eva-Maria; Bondzio, Angelika; Aschenbach, Jörg R; Martens, Holger; Einspanier, Ralf; Scharfen, Franziska; Zentek, Jürgen; Pieper, Robert; Lodemann, Ulrike

    2015-07-01

    After weaning, piglets are often fed diets supplemented with high concentrations of zinc (Zn) to decrease post-weaning diarrhea. The aim of this study was to elucidate the regulation of Zn homeostasis within intestinal epithelial cells during excessive Zn exposure. High Zn concentrations elevated the intracellular Zn level in IPEC-J2 and Caco-2 cells which was influenced by differentiation status and time of exposure. With increasing Zn concentrations, mRNA and protein levels of metallothionein (MT) and zinc transporter 1 (ZnT1) were upregulated, whereas zinc transporter 4 (ZIP4) expression was downregulated. Metal-regulatory transcription factor-1 (MTF1) mRNA expression was upregulated at high Zn concentrations in IPEC-J2 cells, which corresponded to higher intracellular Zn concentrations. Based on these results, we suggest that intestinal epithelial cells adapt the expression of these genes to the amount of extracellular Zn available in order to maintain Zn homeostasis. Cell line-dependent differences in the regulation of Zn homeostasis were detected. PMID:25757458

  17. Omeprazole decreases magnesium transport across Caco-2 monolayers

    Institute of Scientific and Technical Information of China (English)

    Narongrit Thongon; Nateetip Krishnamra

    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 omeprazoletreated 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.

  18. FOXA2 regulates a network of genes involved in critical functions of human intestinal epithelial cells.

    Science.gov (United States)

    Gosalia, Nehal; Yang, Rui; Kerschner, Jenny L; Harris, Ann

    2015-07-01

    The forkhead box A (FOXA) family of pioneer transcription factors is critical for the development of many endoderm-derived tissues. Their importance in regulating biological processes in the lung and liver is extensively characterized, though much less is known about their role in intestine. Here we investigate the contribution of FOXA2 to coordinating intestinal epithelial cell function using postconfluent Caco2 cells, differentiated into an enterocyte-like model. FOXA2 binding sites genome-wide were determined by ChIP-seq and direct targets of the factor were validated by ChIP-qPCR and siRNA-mediated depletion of FOXA1/2 followed by RT-qPCR. Peaks of FOXA2 occupancy were frequent at loci contributing to gene ontology pathways of regulation of cell migration, cell motion, and plasma membrane function. Depletion of both FOXA1 and FOXA2 led to a significant reduction in the expression of multiple transmembrane proteins including ion channels and transporters, which form a network that is essential for maintaining normal ion and solute transport. One of the targets was the adenosine A2B receptor, and reduced receptor mRNA levels were associated with a functional decrease in intracellular cyclic AMP. We also observed that 30% of FOXA2 binding sites contained a GATA motif and that FOXA1/A2 depletion reduced GATA-4, but not GATA-6 protein levels. These data show that FOXA2 plays a pivotal role in regulating intestinal epithelial cell function. Moreover, that the FOXA and GATA families of transcription factors may work cooperatively to regulate gene expression genome-wide in the intestinal epithelium. PMID:25921584

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

    Directory of Open Access Journals (Sweden)

    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

  20. Effect of Mechanical Agitation on Cationic Liposome Transport across an Unstirred Water Layer in Caco-2 Cells.

    Science.gov (United States)

    Kono, Yusuke; Iwasaki, Ayu; Matsuoka, Kenta; Fujita, Takuya

    2016-01-01

    To develop an effective oral delivery system for plasmid DNA (pDNA) using cationic liposomes, it is necessary to clarify the characteristics of uptake and transport of cationic liposome/pDNA complexes into the intestinal epithelium. In particular, evaluation of the involvement of an unstirred water layer (UWL), which is a considerable permeability barrier, in cationic liposome transport is very important. Here, we investigated the effects of a UWL on the transfection efficiency of cationic liposome/pDNA complexes into a Caco-2 cell monolayer. When Caco-2 cells were transfected with cationic liposome/pDNA complexes in shaking cultures to reduce the thickness of the UWL, gene expression was significantly higher in Caco-2 cells compared with static cultures. We also found that this enhancement of gene expression by shaking was not attributable to activation of transcription factors such as activator protein-1 and nuclear factor-kappaB (NF-κB). In addition, the increase in gene expression by mechanical agitation was observed at all charge ratios (1.5, 2.3, 3.1, 4.5) of cationic liposome/pDNA complexes. Transport experiments using Transwells demonstrated that mechanical agitation increased the uptake of cationic liposome/pDNA complexes by Caco-2 cells, whereas transport of the complexes across a Caco-2 cell monolayer did not occurr. Moreover, the augmentation of the gene expression of cationic liposome/pDNA complexes by shaking was observed in Madin-Darby canine kidney cells. These results indicate that a UWL greatly affects the uptake and transfection efficiency of cationic liposome/pDNA complexes into an epithelial monolayer in vitro. PMID:27476939

  1. Effect of hypoxia-mimic deferoxamine on fibroblast growth factor 21 expression in intestine cells%去铁胺对人源肠道 Caco-2细胞成纤维细胞生长因子21表达的影响

    Institute of Scientific and Technical Information of China (English)

    何雪倩; 张翼; 吴玲剑; 陈超; 谢尧琪; 陈李斌佶; 刘彦隆; 林虹; 庞玲霞

    2014-01-01

    Objective To study the effect of hypoxia-mimic deferoxamine ( DFO ) on fibroblast growth factor (FGF21) expression in intestinal Caco-2 cells.Methods Caco-2 cells were either treated with 50, 100, 150, 200μmol/L DFO for 12 h or treated with 200 μmol/L DFO for 4, 8, 12 h.FGF21 mRNA expression were measured by RT-PCR. Caco-2 cells were pretreated with either HIF or ROS inhibitor and then treated with DFO.FGF21 mRNA expression was measured by RT-PCR.Results FGF21 mRNA expression decreased in dose-and time-dependent manners with DFO treat-ment in Caco-2 cells.HIF inhibitor and DFO treatment still decreased FGF21 mRNA expression (all P0.05).Conclusion DFO-induced FGF21 down-regulation is not associated with HIF stabilization, but involves hypoxia-induced oxidative stress.%目的:观察缺氧模拟物去铁胺(DFO)对人源肠道Caco-2细胞成纤维细胞生长因子21(FGF21)表达的影响。方法分别用50、100、150、200μmol/L的DFO处理Caco-2细胞12 h,同时用200μmol/L的DFO处理Caco-2细胞4、8、12 h,RT-PCR法检测细胞中的FGF21 mRNA。分别用缺氧诱导因子( HIF)抑制剂和ROS抑制剂预处理Caco-2细胞,再用DFO继续处理,RT-PCR法检测细胞中的FGF-21 mRNA。结果随DFO浓度增加和作用时间延长,Caco-2细胞FGF21 mRNA相对表达量逐渐降低(P均<0.05)。 HIF抑制剂预处理后,Caco-2细胞FGF21相对表达量依然降低(P均<0.05);ROS抑制剂预处理后,Caco-2细胞FGF21 mRNA相对表达量未出现明显降低(P均>0.05)。结论 DFO对肠道Caco-2细胞FGF21的表达有抑制作用,该作用与缺氧产生HIF无关,而与ROS有关。

  2. Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+ and cell apoptosis in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Li Wang; Qing Li; Xiang-Lin Duan; Yan-Zhong Chang

    2005-01-01

    AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry.RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase Ⅺ and dispase Ⅰ. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control,n = 150) to 35.71±13.99 (n = 150, P<0.01), 72.19±35.40 (n = 150, P<0.01) and 211.34±29.03 (n = 150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 μmol/L). The FIvalue of Caco-2 cells treated by FAC was 185.85±33.77 (n = 150, P<0.01), 122.73±58.47 (n = 150, P<0.01), and 53.29±19.82 (n = 150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10 μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n = 150) to 45.19±21.95 (n = 150, P<0

  3. [Epithelial cell in intestinal homeostasis and inflammatory bowel diseases].

    Science.gov (United States)

    Zouiten-Mekki, Lilia; Serghini, Meriem; Fekih, Monia; Kallel, Lamia; Matri, Samira; Ben Mustapha, Nadia; Boubaker, Jalel; Filali, Azza

    2013-12-01

    Crohn's disease (CD) and ulcerative colitis (UC) are the principal inflammatory bowel diseases (IBD) which physiopathology is currently poorly elucidated. During these diseases, the participation of the epithelial cell in the installation and the perpetuation of the intestinal inflammation is now clearly implicated. In fact, the intestinal epithelium located at the interface between the internal environment and the intestinal luminal, is key to the homeostatic regulation of the intestinal barrier. This barrier can schematically be regarded as being three barriers in one: a physical, chemical and immune barrier. The barrier function of epithelial cell can be altered by various mechanisms as occurs in IBD. The goal of this article is to review the literature on the role of the epithelial cell in intestinal homeostasis and its implication in the IBD. PMID:24356146

  4. Intestinal epithelial vitamin D receptor signaling inhibits experimental colitis

    OpenAIRE

    Liu, Weicheng; Chen, Yunzi; Golan, Maya Aharoni; Annunziata, Maria L.; Du, Jie; Dougherty, Urszula; Kong, Juan; Musch, Mark; Huang, Yong; Pekow, Joel; Zheng, Changqing; Bissonnette, Marc; Hanauer, Stephen B.; Li, Yan Chun

    2013-01-01

    The inhibitory effects of vitamin D on colitis have been previously documented. Global vitamin D receptor (VDR) deletion exaggerates colitis, but the relative anticolitic contribution of epithelial and nonepithelial VDR signaling is unknown. Here, we showed that colonic epithelial VDR expression was substantially reduced in patients with Crohn’s disease or ulcerative colitis. Moreover, targeted expression of human VDR (hVDR) in intestinal epithelial cells (IECs) protected mice from developing...

  5. Coating with luminal gut-constituents alters adherence of nanoparticles to intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Heike Sinnecker

    2014-12-01

    Full Text Available Background: Anthropogenic nanoparticles (NPs have found their way into many goods of everyday life. Inhalation, ingestion and skin contact are potential routes for NPs to enter the body. In particular the digestive tract with its huge absorptive surface area provides a prime gateway for NP uptake. Considering that NPs are covered by luminal gut-constituents en route through the gastrointestinal tract, we wanted to know if such modifications have an influence on the interaction between NPs and enterocytes.Results: We investigated the consequences of a treatment with various luminal gut-constituents on the adherence of nanoparticles to intestinal epithelial cells. Carboxylated polystyrene particles 20, 100 and 200 nm in size represented our anthropogenic NPs, and differentiated Caco-2 cells served as model for mature enterocytes of the small intestine. Pretreatment with the proteins BSA and casein consistently reduced the adherence of all NPs to the cultured enterocytes, while incubation of NPs with meat extract had no obvious effect on particle adherence. In contrast, contact with intestinal fluid appeared to increase the particle-cell interaction of 20 and 100 nm NPs.Conclusion: Luminal gut-constituents may both attenuate and augment the adherence of NPs to cell surfaces. These effects appear to be dependent on the particle size as well as on the type of interacting protein. While some proteins will rather passivate particles towards cell attachment, possibly by increasing colloid stability or camouflaging attachment sites, certain components of intestinal fluid are capable to modify particle surfaces in such a way that interactions with cellular surface structures result in an increased binding.

  6. Calcium-Ask1-MKK7-JNK2-c-Src Signaling Cascade Mediates Disruption of Intestinal Epithelial Tight Junctions by Dextran Sulfate Sodium

    Science.gov (United States)

    Samak, Geetha; Chaudhry, Kamaljit K.; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H.; Rao, RadhaKrishna

    2015-01-01

    Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with the symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca2+ concentration, and depletion of intracellular Ca2+ by BAPTA or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of Ask1 or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased Tyr-phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. SP600125 abrogated DSS-induced Tyr-phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto phosphorylation of c-Src. This study demonstrates that Ca2+-Ask1-MKK7-JNK2-cSrc signaling cascade mediates DSS-induced tight junction disruption and barrier dysfunction. PMID:25377781

  7. Degradation of coeliac disease-inducing rye secalin by germinating cereal enzymes: diminishing toxic effects in intestinal epithelial cells.

    Science.gov (United States)

    Stenman, S M; Lindfors, K; Venäläinen, J I; Hautala, A; Männistö, P T; Garcia-Horsman, J A; Kaukovirta-Norja, A; Auriola, S; Mauriala, T; Mäki, M; Kaukinen, K

    2010-08-01

    Currently the only treatment for coeliac disease is a lifelong gluten-free diet excluding food products containing wheat, rye and barley. There is, however, only scarce evidence as to harmful effects of rye in coeliac disease. To confirm the assumption that rye should be excluded from the coeliac patient's diet, we now sought to establish whether rye secalin activates toxic reactions in vitro in intestinal epithelial cell models as extensively as wheat gliadin. Further, we investigated the efficacy of germinating cereal enzymes from oat, wheat and barley to hydrolyse secalin into short fragments and whether secalin-induced harmful effects can be reduced by such pretreatment. In the current study, secalin elicited toxic reactions in intestinal Caco-2 epithelial cells similarly to gliadin: it induced epithelial cell layer permeability, tight junctional protein occludin and ZO-1 distortion and actin reorganization. In high-performance liquid chromatography and mass spectroscopy (HPLC-MS), germinating barley enzymes provided the most efficient degradation of secalin and gliadin peptides and was thus selected for further in vitro analysis. After germinating barley enzyme pretreatment, all toxic reactions induced by secalin were ameliorated. We conclude that germinating enzymes from barley are particularly efficient in the degradation of rye secalin. In future, these enzymes might be utilized as a novel medical treatment for coeliac disease or in food processing in order to develop high-quality coeliac-safe food products. PMID:20560983

  8. 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. PMID:24746839

  9. Regulatory effect of heat shock protein 70 in stress-induced rat intestinal epithelial barrier dysfunction

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    Ping-Chang Yang

    2009-01-01

    Full Text Available Background : Psychological stress is one of the factors associated with many human diseases; the mechanisms need to be further understood. Methods : Rats were subjected to chronic water avoid stress. Intestinal epithelial heat shock protein (HSP 70 was evaluated. The intestinal epithelial permeability was examined with Ussing chamber technique. Results : HSP70 was detected in normal intestinal epithelial cells. Psychological stress decreased HSP70 in the intestinal epithelial cells that correlated with the stress-induced intestinal epithelial hyperpermeability. Pretreatment with HSP70 abrogated stress-induced intestinal barrier dysfunction. Conclusions : Chronic stress inhibits HSP70 activity in rat intestinal epithelial layer that is associated with intestinal epithelial barrier dysfunction, which can be prevented by pretreatment with HSP70 protein. (Yang PC, Tu YH, Perdue MH, Oluwole C, Struiksma S. Regulatory effect of heat shock protein 70 in stress-induced rat intestinal epithelial barrier dysfunction.

  10. The viability and intestinal epithelial cell adhesion of probiotic strain combination--in vitro study.

    Science.gov (United States)

    Piątek, Jacek; Gibas-Dorna, Magdalena; Olejnik, Anna; Krauss, Hanna; Wierzbicki, Krzysztof; Żukiewicz-Sobczak, Wioletta; Głowacki, Maciej

    2012-01-01

    To be effective, probiotic bacteria must exhibit a number of functional characteristics, including the resistance to gastric acidity and the ability to adhere to the intestinal epithelium. In this study, we examined in vitro the viability of lactic acid bacteria (LAB) combination after exposure to low pH, and the adhesion of LAB to Caco-2 cells during coincubation of 9 bacterial strains. To test bacterial viability, 6 commercially available products were incubated in 0.1 N HCl at pH 1.2 for 60 min. The greatest growth inhibition was noted for the non-capsulated product containing the Lactobacillus rhamnosus strain (log reduction of CFU = 6.4), and the best survival observed for the product containing 9 bacterial strains, equipped with a modern capsule made according to the Multi-Resistant Encapsulation technology (log reduction of CFU = 0.1). In the adhesion experiment, the combination of 9 bacterial strains was added to 17-day-old Caco-2 cell culture for 90 min. The greatest efficiency of adhesion was observed for the inoculum containing 5.5x10(8) CFU/mL/9.6 cm(2) of Caco-2 and the dose of probiotic bacteria of 190 cells per one Caco-2 cell. As a result, approximately 157 bacterial cells adhered to one Caco-2 cell. The results indicate that the combination of 9 bacterial strains in the examined product is characterized as highly adhesive. PMID:22462453

  11. Immune-epithelial crosstalk at the intestinal surface.

    Science.gov (United States)

    Wittkopf, Nadine; Neurath, Markus F; Becker, Christoph

    2014-03-01

    The intestinal tract is one of the most complex organs of the human body. It has to exercise various functions including food and water absorption, as well as barrier and immune regulation. These functions affect not only the gut itself, but influence the overall health of the organism. Diseases involving the gastrointestinal tract such as inflammatory bowel disease and colorectal cancer therefore severely affect the patient's quality of life and can become life-threatening. Intestinal epithelial cells (IECs) play an important role in intestinal inflammation, infection, and cancer development. IECs not only constitute the first barrier in the gut against the lumen, they also constantly signal information about the gut lumen to immune cells, thereby influencing their behaviour. In contrast, by producing various antimicrobial peptides, IECs shape the microbial community within the gut. IECs also respond to cytokines and other mediators of immune cells in the lamina propria. Interactions between epithelial cells and immune cells in the intestine are responsible for gut homeostasis, and modulations of this crosstalk have been reported in studies of gut diseases. This review discusses the wide field of immune-epithelial interactions and shows the importance of immune-epithelial crosstalk in the intestine to gut homeostasis and the overall health status. PMID:24469679

  12. Attachment of Giardia lamblia to rat intestinal epithelial cells.

    OpenAIRE

    Inge, P M; Edson, C M; Farthing, M J

    1988-01-01

    The human enteric protozoan, Giardia lamblia, has surface membrane lectin activity which mediates parasite adherence to erythrocytes. To determine whether an intestinal binding site exists for this lectin we have studied the interaction in vitro between axenically cultured Giardia trophozoites and isolated rat intestinal epithelial cells. Scanning electron microscopy showed that Giardia attached to the apical microvillus membrane and basolateral membrane of rat enterocytes. Any location on th...

  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. KLF4 regulation in intestinal epithelial cell maturation

    International Nuclear Information System (INIS)

    The Krueppel-like factor 4 (KLF4) transcription factor suppresses tumorigenesis in gastrointestinal epithelium. Thus, its expression is decreased in gastric and colon cancers. Moreover, KLF4 regulates both differentiation and growth that is likely fundamental to its tumor suppressor activity. We dissected the expression of Klf4 in the normal mouse intestinal epithelium along the crypt-villus and cephalo-caudal axes. Klf4 reached its highest level in differentiated cells of the villus, with levels in the duodenum > jejunum > ileum, in inverse relation to the representation of goblet cells in these regions, the lineage previously linked to KLF4. In parallel, in vitro studies using HT29cl.16E and Caco2 colon cancer cell lines clarified that KLF4 increased coincident with differentiation along both the goblet and absorptive cell lineages, respectively, and that KLF4 levels also increased during differentiation induced by the short chain fatty acid butyrate, independently of cell fate. Moreover, we determined that lower levels of KLF4 expression in the proliferative compartment of the intestinal epithelium are regulated by the transcription factors TCF4 and SOX9, an effector and a target, respectively, of β-catenin/Tcf signaling, and independently of CDX2. Thus, reduced levels of KLF4 tumor suppressor activity in colon tumors may be driven by elevated β-catenin/Tcf signaling

  15. Butyrate produced by commensal bacteria potentiates phorbol esters induced AP-1 response in human intestinal epithelial cells.

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    Malgorzata Nepelska

    Full Text Available The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect of culture supernatant from 49 commensal strains. We observed that several bacteria were able to activate the AP-1 pathway and this was correlated to the amount of short chain fatty acids (SCFAs produced. Besides being a major source of energy for epithelial cells, SCFAs have been shown to regulate several signaling pathways in these cells. We show that propionate and butyrate are potent activators of the AP-1 pathway, butyrate being the more efficient of the two. We also observed a strong synergistic activation of AP-1 pathway when using butyrate with PMA, a PKC activator. Moreover, butyrate enhanced the PMA-induced expression of c-fos and ERK1/2 phosphorylation, but not p38 and JNK. In conclusion, we showed that SCFAs especially butyrate regulate the AP-1 signaling pathway, a feature that may contribute to the physiological impact of the gut microbiota on the host. Our results provide support for the involvement of butyrate in modulating the action of PKC in colon cancer cells.

  16. Sugars increase non-heme iron bioavailability in human epithelial intestinal and liver cells.

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    Tatiana Christides

    Full Text Available Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55 increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl3 increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.

  17. Regulatory effect of heat shock protein 70 in stress-induced rat intestinal epithelial barrier dysfunction

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    Stevie Struiksma

    2009-06-01

    Full Text Available Background: Psychological stress is one of the factors associated with many human diseases; the mechanisms need to be further understood. Methods: Rats were subjected to chronic water avoid stress. Intestinal epithelial heat shock protein (HSP 70 was evaluated. The intestinal epithelial permeability was examined with Ussing chamber technique. Results: HSP70 was detected in normal intestinal epithelial cells. Psychological stress decreased HSP70 in the intestinal epithelial cells that correlated with the stress-induced intestinal epithelial hyperpermeability. Pretreatment with HSP70 abrogated stress-induced intestinal barrier dysfunction. Conclusions: Chronic stress inhibits HSP70 activity in rat intestinal epithelial layer that is associated with intestinal epithelial barrier dysfunction, which can be prevented by pretreatment with HSP70 protein.

  18. Epigenetics in Intestinal Epithelial Cell Renewal.

    Science.gov (United States)

    Roostaee, Alireza; Benoit, Yannick D; Boudjadi, Salah; Beaulieu, Jean-François

    2016-11-01

    A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt-villus axis. One important check-point in this process occurs in the transit amplifying zone of the intestinal crypts where different signaling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development, and tissue renewal. J. Cell. Physiol. 231: 2361-2367, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:27061836

  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

    2016-05-01

    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. 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.

  1. 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.

  2. Fish Oil Enhances Recovery of Intestinal Microbiota and Epithelial Integrity in Chronic Rejection of Intestinal Transplant

    Science.gov (United States)

    Li, Qiurong; Zhang, Qiang; Wang, Chenyang; Tang, Chun; Zhang, Yanmei; Li, Ning; Li, Jieshou

    2011-01-01

    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. PMID:21698145

  3. Genetically Modified Caco-2 Cells With Improved Cytochrome P450 Metabolic Capacity.

    Science.gov (United States)

    Küblbeck, Jenni; Hakkarainen, Jenni J; Petsalo, Aleksanteri; Vellonen, Kati-Sisko; Tolonen, Ari; Reponen, Petri; Forsberg, Markus M; Honkakoski, Paavo

    2016-02-01

    The human intestinal Caco-2 cell line has been extensively used as a model of small intestinal absorption but it lacks expression and function of cytochrome P450 enzymes, particularly CYP3A4 and CYP2C9, which are normally expressed in the intestinal epithelium. In order to increase the expression and activity of CYP isozymes in these cells, we created 2 novel Caco-2 sublines expressing chimeric constitutive androstane or pregnane X receptors and characterized these cells for their metabolic and absorption properties. In spite of elevated mRNA expression of transporters and differentiation markers, the permeation properties of the modified cell lines did not significantly differ from those of the wild-type cells. In contrast, the metabolic activity was increased beyond the currently used models. Specifically, CYP3A4 activity was increased up to 20-fold as compared to vitamin D treated wild-type Caco-2 cells. PMID:26869438

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

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    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

  5. Alternative Functional In Vitro Models of Human Intestinal Epithelia

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    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.

  6. Uptake of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) and dicamba (3,6-dichloro-2-methoxybenzoic acid) from the apical membranes of the human intestinal Caco-2 cells.

    Science.gov (United States)

    Kimura, Osamu; Tsukagoshi, Kensuke; Hayasaka, Moriaki; Endo, Tetsuya

    2012-01-01

    We investigated whether the uptake of triclopyr (3, 5, 6-trichloro-2-pyridinyloxyacetic acid) and dicamba (3,6-dichloro-2-methoxybenzoic acid) across the apical membrane of Caco-2 cells was mediated via proton-linked monocarboxylic acid transporters (MCTs). The uptake of triclopyr from the apical membranes was fast, pH-, temperature-, and concentration dependent, required metabolic energy to proceed, and was competitively inhibited by monocarboxylic acids such as benzoic acid and ferulic acid (substrates of L-lactic acid-insensitive MCTs), but not by L-lactic acid. Thus, the uptake of triclopyr in Caco-2 cells appears to be mediated mainly via L-lactic acid-insensitive MCTs. In contrast, the uptake of dicamba (a benzoic acid derivative) was slow, and it was both pH- and temperature dependent. Coincubation with ferulic acid did not decrease the uptake of dicamba, although coincubation with benzoic acid moderately decreased it. The uptake of dicamba appears to be mediated mainly via passive diffusion, which is in contrast to the uptake of benzoic acid via MCTs. We speculate that the substituted groups in dicamba may inhibit uptake via MCTs. PMID:21766207

  7. Labile complexes facilitate cadmium uptake by Caco-2 cells

    International Nuclear Information System (INIS)

    The Free Ion Activity Model (FIAM) predicts that metal uptake in biota is related to the free ion activity in the external solution and that metal complexes do not contribute. However, studies with plants have shown that labile metal complexes enhance metal bioavailability when the uptake is rate-limited by transport of the free ion in solution to the uptake site. Here, the role of labile complexes of Cd on metal bioavailability was assessed using Caco-2 cells, the cell model for intestinal absorption. At low Cd2+ concentration (1 nM), the CdCln2−n complexes contributed to the uptake almost to the same extent as the free ion. At large Cd2+ concentration (10 μM), the contribution of the complexes was much smaller. At constant Cd2+ concentration, Cd intake in the cells from solutions containing synthetic ligands such as EDTA increased as the dissociation rate of the cadmium complexes increased, and correlated well with the Cd diffusion flux in solution measured with the Diffusive Gradient in Thin Films technique (DGT). The Cd intake fluxes in the cells were well predicted assuming that the specific uptake is limited by diffusion of the free Cd2+ ion to the cell surface. Our results underline that speciation of Cd has a major effect on its uptake by intestinal cells, but the availability is not simply related to the free ion concentration. Labile complexes of Cd enhance metal bioavailability in these cells, likely by alleviating diffusive limitations. - Highlights: ► We examined the role of labile complexes on the uptake of Cd and Zn by Caco-2 cells. ► The availability of Cd and Zn is not simply related to the free ion activity. ► Labile complexes of Cd enhance metal bioavailability in the Caco-2 cells at low free ion activities. ► The active uptake is limited by diffusion of the free Cd2+ ion to the cell surface.

  8. Polarizing intestinal epithelial cells electrically through Ror2

    OpenAIRE

    Cao, L; McCaig, CD; Scott, RH; Zhao, S.; G. Milne; Clevers, H; Zhao, M; Pu, J

    2014-01-01

    ABSTRACT The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here, we show that applying a physiological electric field to intestinal epithelial cells, to mimic the natural electric field created by the transepithelial potential difference, polarized p...

  9. An experimental platform using human intestinal epithelial cell lines to differentiate between hazardous and non-hazardous proteins.

    Science.gov (United States)

    Hurley, Bryan P; Pirzai, Waheed; Eaton, Alex D; Harper, Marc; Roper, Jason; Zimmermann, Cindi; Ladics, Gregory S; Layton, Raymond J; Delaney, Bryan

    2016-06-01

    Human intestinal epithelial cell lines (T84, Caco-2, and HCT-8) grown on permeable Transwell™ filters serve as models of the gastrointestinal barrier. In this study, this in vitro model system was evaluated for effectiveness at distinguishing between hazardous and non-hazardous proteins. Indicators of cytotoxicity (LDH release, MTT conversion), monolayer barrier integrity ([(3)H]-inulin flux, horseradish peroxidase flux, trans-epithelial electrical resistance [TEER]), and inflammation (IL-8, IL-6 release) were monitored following exposure to hazardous or non-hazardous proteins. The hazardous proteins examined include streptolysin O (from Streptococcus pyogenes), Clostridium difficile Toxins A and B, heat-labile toxin from enterotoxigenic Escherichia coli, listeriolysin O (from Listeria monocytogenes), melittin (from bee venom), and mastoparan (from wasp venom). Non-hazardous proteins included bovine and porcine serum albumin, bovine fibronectin, and ribulose bisphosphate carboxylase/oxygenase (RuBisco) from spinach. Food allergenic proteins bovine milk β-lactoglobulin and peanut Ara h 2 were also tested as was the anti-nutritive food protein wheat germ agglutinin. Results demonstrated that this model system effectively distinguished between hazardous and non-hazardous proteins through combined analysis of multiple cells lines and assays. This experimental strategy may represent a useful adjunct to multi-component analysis of proteins with unknown hazard profiles. PMID:27060235

  10. Modulation of Intestinal Epithelial Defense Responses by Probiotic Bacteria.

    Science.gov (United States)

    Wan, L Y M; Chen, Z J; Shah, N P; El-Nezami, H

    2016-12-01

    Probiotics are live microorganisms, which when administered in food confer numerous health benefits. In previous studies about beneficial effects of probiotic bacteria to health, particularly in the fields of intestinal mucosa defense responses, specific probiotics, in a strain-dependent manner, show certain degree of potential to reinforce the integrity of intestinal epithelium and/or regulate some immune components. The mechanism of probiotic action is an area of interest. Among all possible routes of modulation by probiotics of intestinal epithelial cell-mediated defense responses, modulations of intestinal barrier function, innate, and adaptive mucosal immune responses, as well as signaling pathways are considered to play important role in the intestinal defense responses against pathogenic bacteria. This review summarizes the beneficial effects of probiotic bacteria to intestinal health together with the mechanisms affected by probiotic bacteria: barrier function, innate, and adaptive defense responses such as secretion of mucins, defensins, trefoil factors, immunoglobulin A (IgA), Toll-like receptors (TLRs), cytokines, gut associated lymphoid tissues, and signaling pathways. PMID:25629818

  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. 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. PMID:25651187

  13. 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. PMID:26795356

  14. Interaction exists between matriptase inhibitors and intestinal epithelial cells.

    Science.gov (United States)

    Pászti-Gere, Erzsebet; Barna, Réka Fanni; Ujhelyi, Gabriella; Steinmetzer, Torsten

    2016-10-01

    The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro. PMID:26118419

  15. 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...

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

    International Nuclear Information System (INIS)

    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

  17. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kimmich, G.A.

    1990-01-01

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

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

    International Nuclear Information System (INIS)

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

  20. 1,25-Dihydroxyvitamin D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated MLCK-P-MLC signaling pathway.

    Science.gov (United States)

    Chen, Shanwen; Zhu, Jing; Chen, Guowei; Zuo, Shuai; Zhang, Junling; Chen, Ziyi; Wang, Xin; Li, Junxia; Liu, Yucun; Wang, Pengyuan

    2015-05-01

    Substantial studies have demonstrated the protective effect of 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) on intestinal barrier function, but the mechanisms are not fully illustrated. In this study, the effect of 1,25(OH)2D3 on TNF-α induced barrier dysfunction was further investigated in Caco-2 cell monolayers. The barrier function of Caco-2 monolayers was evaluated by measuring trans-epithelial electrical resistance (TEER) and FITC-Dextran 40,000 Da (FD-40) trans-membrane flux. ZO-1 and Occludin were chosen as markers of the localization of tight junction (TJ) proteins for immunofluorescence. The expression of MLCK and phosphorylation level of myosin light chain (MLC) were measured by immunoblotting. The activation of NF-kB p65 was analyzed by EMSA and immunofluorescence. The results suggest that 1,25(OH)2D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated activation of MLCK-P-MLC signaling pathway. PMID:25838204

  1. Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells.

    Science.gov (United States)

    Eaves-Pyles, Tonyia; Allen, Christopher A; Taormina, Joanna; Swidsinski, Alexander; Tutt, Christopher B; Jezek, G Eric; Islas-Islas, Martha; Torres, Alfredo G

    2008-07-01

    Inflammatory diseases of the intestinal tract are a major health concern both in the United States and around the world. Evidence now suggests that a new category of Escherichia coli, designated Adherent Invasive E. coli (AIEC) is highly prevalent in Crohn's Disease (CD) patients. AIEC strains have been shown to colonize and adhere to intestinal epithelial cells (IEC). However, the role AIEC strains play in the induction of an inflammatory response is not known. Therefore, we examined several E. coli strains (designated LF82, O83:H1, 6604 and 6655) that were isolated from CD patients for their ability to induce inflammation in two IEC, Caco-2BBe and T-84 cells. Results showed that each strain had varying abilities to adhere to and invade IEC as well as induced cytokine secretion from polarized IEC. However, E. coli O83:H1 displayed the best characteristics of AIEC strains as compared to the prototype AIEC strain LF82, inducing cytokine secretion from IEC and promoting immune cell migration through IEC. Upon further analysis, E. coli O83:H1 did not harbor virulence genes present in known pathogenic intestinal organisms. Further characterization of E. coli O83:H1 virulence determinants showed that a non-flagellated O83:H1 strain significantly decreased the organism's ability to adhere to and invade both IEC and elicit IEC cytokine secretion compared to the wild type and complemented strains. These findings demonstrate that E. coli O83:H1 possesses the characteristics of the AIEC LF82 strain that may contribute to the low-grade, chronic inflammation observed in Crohn's disease. PMID:17900983

  2. 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

    2013-01-01

    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. PMID:24192850

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

    International Nuclear Information System (INIS)

    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 (R2 = 0.92) to the human oral adsorption than that of the Transwell culture (R2 = 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. (paper)

  4. Apical Gene Transfer into Quiescent Human and Canine Polarized Intestinal Epithelial Cells by Lentivirus Vectors

    OpenAIRE

    Seppen, Jurgen; Barry, Simon C.; Klinkspoor, J. Henriette; Katen, Louis J.; Lee, Sum P; Garcia, J. Victor; Osborne, William R. A.

    2000-01-01

    Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.

  5. Interleukin-23 Increases Intestinal Epithelial Cell Permeability In Vitro.

    Science.gov (United States)

    Heinzerling, Nathan P; Donohoe, Deborah; Fredrich, Katherine; Gourlay, David M; Liedel, Jennifer L

    2016-06-01

    Background Breast milk has a heterogeneous composition that differs between mothers and changes throughout the first weeks after birth. The proinflammatory cytokine IL-23 has a highly variable expression in human breast milk. We hypothesize that IL-23 found in human breast milk is biologically active and promotes epithelial barrier dysfunction. Methods The immature rat small intestinal epithelial cell line, IEC-18, was grown on cell inserts or standard cell culture plates. Confluent cultures were exposed to human breast milk with high or low levels of IL-23 and barrier function was measured using a flux of fluorescein isothiocyanate-dextran (FD-70). In addition, protein and mRNA expression of occludin and ZO-1 were measured and immunofluorescence used to stain occludin and ZO-1. Results Exposure to breast milk with high levels of IL-23 caused an increase flux of FD-70 compared with both controls and breast milk with low levels of IL-23. The protein expression of ZO-1 but not occludin was decreased by exposure to high levels of IL-23. These results correlate with immunofluorescent staining of ZO-1 and occludin which show decreased staining of occludin in both the groups exposed to breast milk with high and low IL-23. Conversely, cells exposed to high IL-23 breast milk had little peripheral staining of ZO-1 compared with controls and low IL-23 breast milk. Conclusion IL-23 in human breast milk is biologically active and negatively affects the barrier function of intestinal epithelial cells through the degradation of tight junction proteins. PMID:26007691

  6. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen Lillelund;

    2002-01-01

    The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change in the...... a general down-regulation of genes in the low abundance class. Similar results were found using mouse small intestinal crypt and villus cells, suggesting that the phenomenon also occurs in the intestine in vivo. The expression data were subsequently used in a search for markers for subsets of...... epithelial cells by performing reverse transcriptase-polymerase chain reaction on RNA extracted from laser dissected intestinal crypt and villi. In a screen of eight transcripts one - SART3 - was identified as a marker for human colonic crypts....

  7. Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures

    Directory of Open Access Journals (Sweden)

    Mainil Jacques

    2005-12-01

    Full Text Available Abstract Background Cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract. Results Cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein as well as an epithelial cytoskeleton component (cytokeratin 18. However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker. Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1–2 mM or using a glucose-deprived culture medium. Conclusion The present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations.

  8. Acetaminophen Changes Intestinal Epithelial Cell Membrane Properties, Subsequently Affecting Absorption Processes

    Directory of Open Access Journals (Sweden)

    Christine Schäfer

    2013-08-01

    Full Text Available Background/Aims: Acetaminophen (APAP effects on intestinal barrier properties are less investigated. APAP may lead to a changed bioavailability of a subsequently administered drug or diet in the body. We investigated the influence of APAP on enterocytic cell membrane properties that are able to modify the net intestinal absorption of administered substances across the Caco-2 barrier model. Methods: The effect of APAP on cytotoxicity was measured by LDH assay, TER value and cell capacitance label-free using impedance monitoring, membrane permeability by FITC-dextrans, and efflux transporter MDR1 activity by Rh123. APAP levels were determined by HPLC analysis. Cell membrane topography and microvilli were investigated using SEM and intestinal alkaline phosphatase (Alpi and tight junction protein 1 (TJP1 expression by western blot analysis. Results: APAP changed the apical cell surface, reduced the number of microvilli and protein expression of Alpi as a brush border marker and TJP1, increased the membrane integrity and concurrently decreased cell capacitance over time. In addition, APAP decreased the permeability to small molecules and increased the efflux transporter activity, MDR1. Conclusion: APAP alters the Caco-2 cell membrane properties by different mechanisms and reduces the permeability to administered substances. These findings may help to optimize therapeutic implications.

  9. Bovine lactoferrin regulates cell survival, apoptosis and inflammation in intestinal epithelial cells and preterm pig intestine

    DEFF Research Database (Denmark)

    Nguyen, Duc Ninh; Jiang, Pingping; Stensballe, Allan; Bendixen, Emøke; Sangild, Per T.; Chatterton, Dereck E. W.

    2016-01-01

    Bovine lactoferrin (bLF) may modulate neonatal intestinal inflammation. Previous studies in intestinal epithelial cells (IECs) indicated that moderate bLF doses enhance proliferation whereas high doses trigger inflammation. To further elucidate cellular mechanisms, we profiled the porcine IEC...... proteome after stimulation with bLF at 0, 0.1, 1 and 10g/L by LC-MS-based proteomics. Key pathways were analyzed in the intestine of formula-fed preterm pigs with and without supplementation of 10g/L bLF. Levels of 123 IEC proteins were altered by bLF. Low bLF doses (0.1-1g/L) up-regulated 11 proteins...... acid metabolism, and altered three proteins enhancing the hypoxia inducible factor-1 (HIF-1) pathway. In the preterm pig intestine, bLF at 10g/L decreased villus height/crypt depth ratio and up-regulated the Bax/Bcl-2 ratio and HIF-1α, indicating elevated intestinal apoptosis and inflammation. In...

  10. Critical Roles of Intestinal Epithelial Vitamin D Receptor Signaling in Controlling Gut Mucosal Inflammation

    OpenAIRE

    Li, Yan Chun; Chen, Yunzi; Du, Jie

    2015-01-01

    Although vitamin D receptor (VDR) is highly expressed in the intestine, the role of VDR signaling in the gut is not fully understood. Our recent studies unveil a regulatory circuit that centers gut epithelial VDR as a key molecule in the control of mucosal inflammation and colitis development. On the one hand, intestinal epithelial VDR signaling protects the integrity of the mucosal barrier by inhibiting inflammation-induced epithelial cell apoptosis. This barrier-protecting, anti-colitic act...

  11. Campylobacter jejuni outer membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin.

    Science.gov (United States)

    Elmi, Abdi; Nasher, Fauzy; Jagatia, Heena; Gundogdu, Ozan; Bajaj-Elliott, Mona; Wren, Brendan; Dorrell, Nick

    2016-04-01

    Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram-negative bacteria. Campylobacter jejuni produces OMVs that trigger IL-8, IL-6, hBD-3 and TNF-α responses from T84 intestinal epithelial cells and are cytotoxic to Caco-2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E-cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co-incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E-cadherin and occludin. The addition of 11168H OMVs to the co-culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time-dependent and dose-dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells. PMID:26451973

  12. The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans.

    Science.gov (United States)

    Ludington, Jacob G; Ward, Honorine D

    2016-05-01

    The apicomplexan parasite Cryptosporidium causes significant diarrheal disease worldwide. Effective anticryptosporidial agents are lacking, in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood. Previously, we identified and characterized a novel Cryptosporidium parvum C-type lectin domain-containing mucin-like glycoprotein, CpClec. In this study, we evaluated the mechanisms underlying interactions of CpClec with intestinal epithelial cells by using an Fc-tagged recombinant protein. CpClec-Fc displayed Ca(2+)-dependent, saturable binding to HCT-8 and Caco-2 cells and competitively inhibited C. parvum attachment to and infection of HCT-8 cells. Binding of CpClec-Fc was specifically inhibited by sulfated glycosaminoglycans, particularly heparin and heparan sulfate. Binding was reduced after the removal of heparan sulfate and following the inhibition of glycosaminoglycan synthesis or sulfation in HCT-8 cells. Like CpClec-Fc binding, C. parvum attachment to and infection of HCT-8 cells were inhibited by glycosaminoglycans and were reduced after heparan sulfate removal or inhibition of glycosaminoglycan synthesis or sulfation. Lastly, CpClec-Fc binding and C. parvum sporozoite attachment were significantly decreased in CHO cell mutants defective in glycosaminoglycan synthesis. Together, these results indicate that CpClec is a novel C-type lectin that mediates C. parvum attachment and infection via Ca(2+)-dependent binding to sulfated proteoglycans on intestinal epithelial cells. PMID:26975991

  13. The role of intestinal epithelial barrier function in the development of NEC

    OpenAIRE

    Halpern, Melissa D.; Patricia W Denning

    2015-01-01

    The intestinal epithelial barrier plays an important role in maintaining host health. Breakdown of intestinal barrier function is known to play a role in many diseases such as infectious enteritis, idiopathic inflammatory bowel disease, and neonatal inflammatory bowel diseases. Recently, increasing research has demonstrated the importance of understanding how intestinal epithelial barrier function develops in the premature neonate in order to develop strategies to promote its maturation. Opti...

  14. 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.

  15. 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.

  16. 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.

  17. Transcription of the Tollip gene is elevated in intestinal epithelial cells through impaired O-GlcNAcylation-dependent nuclear translocation of the negative regulator Elf-1

    International Nuclear Information System (INIS)

    Highlights: → Transcriptional activation of the Tollitip gene is higher in IECs than in monocytes. → Nt -194/-186 region acts as a cis-element and is recognized by Elf-1. → Elf-1 suppresses Tollip gene transcription in monocytes but not in IECs. → O-GlcNAc modification is necessary for nuclear translocation of Elf-1. → O-GlcNAcylation-dependent nuclear translocation of Elf-1 is impaired in IECs. -- Abstract: Intestinal epithelial cells (IECs) must be tolerant of the large number of commensal bacteria inhabiting the intestinal tract to avoid excessive inflammatory reactions. Toll-interacting protein (Tollip), a negative regulator of Toll-like receptor signaling, is known to be expressed at high levels in IECs, and to thereby contribute to the hyporesponsiveness of IECs to commensals. In this study, we analyzed the underlying mechanisms for elevated transcription of the Tollip gene in IECs using a human IEC line, Caco-2, and a human monocyte line, THP-1, as a control. Elf-1 was identified as a transcription factor that negatively regulates Tollip gene expression. The transcription factor Elf-1 was localized in the nucleus by O-linked N-acetylglucosamine (O-GlcNAc) modification, whereas the unmodified form was detected only in the cytoplasm. Comparison of Caco-2 and THP-1 cells revealed that O-GlcNAc modification of Elf-1 was significantly lower in IECs than in monocytes. Collectively, the results indicate that insufficient O-GlcNAc modification prevents Elf-1-mediated transcriptional repression and thereby upregulates Tollip gene expression in IECs.

  18. Effect of Dachengqi Tang (大承气汤) Granule on Proliferation of Intestinal Epithelial Cells in Rats with Experimental Intestinal Obstruction

    Institute of Scientific and Technical Information of China (English)

    康毅; 林秀珍

    2003-01-01

    Objective: To study the effects of Dachengqi Tang (DCQT) granule on the proliferation of the intestinal epithelial cells in rats with experimental intestinal obstruction. Methods: Experimental intestinal obstruction models were established in rats and autoradiography with 3H-TdR was used to determine 3H-TdR labeling counts of intestinal epithelial cells in rats. Results: DCQT granule had no effects on 3H-TdR labeling counts of intestinal epithelial cells in normal rats. DCQT granule obviously increases the rate of renovation in intestinal epithelial cells of the intestinal obstruction rats. Conclusion: DCQT granule could reinforce the intestinal mucosa's defensive function by means of increasing the proliferation of intestinal epithelial cells.

  19. Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

    OpenAIRE

    Finamore, Alberto; Roselli, Marianna; Imbinto, Ambra; Seeboth, Julie; Oswald, Isabelle P.

    2014-01-01

    Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698(T), a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infe...

  20. Toxicity of food-relevant nanoparticles in intestinal epithelial models

    Science.gov (United States)

    McCracken, Christie

    Nanoparticles are increasingly being incorporated into common consumer products, including in foods and food packaging, for their unique properties at the nanoscale. Food-grade silica and titania are used as anti-caking and whitening agents, respectively, and these particle size distributions are composed of approximately one-third nanoparticles. Zinc oxide and silver nanoparticles can be used for their antimicrobial properties. However, little is known about the interactions of nanoparticles in the body upon ingestion. This study was performed to investigate the role of nanoparticle characteristics including surface chemistry, dissolution, and material type on toxicity to the intestinal epithelium. Only mild acute toxicity of zinc oxide nanoparticles was observed after 24-hour treatment of intestinal epithelial C2BBe1 cells based on the results of toxicity assays measuring necrosis, apoptosis, membrane damage, and mitochondrial activity. Silica and titanium dioxide nanoparticles were not observed to be toxic although all nanoparticles were internalized by cells. In vitro digestion of nanoparticles in solutions representing the stomach and intestines prior to treatment of cells did not alter nanoparticle toxicity. Long-term repeated treatment of cells weekly for 24 hours with nanoparticles did not change nanoparticle cytotoxicity or the growth rate of the treated cell populations. Thus, silica, titanium dioxide, and zinc oxide nanoparticles were found to induce little toxicity in intestinal epithelial cells. Fluorescent silica nanoparticles were synthesized as a model for silica used in foods that could be tracked in vitro and in vivo. To maintain an exterior of pure silica, a silica shell was hydrolyzed around a core particle of quantum dots or a fluorescent dye electrostatically associated with a commercial silica particle. The quantum dots used were optimized from a previously reported microwave quantum dot synthesis to a quantum yield of 40%. Characterization

  1. 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.

  2. Action of cholera toxin in the intestinal epithelial cells

    International Nuclear Information System (INIS)

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with a large number of high affinity binding sites in the cell membrane. Binding of 125I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected. The response (elevation of cellular cAMP) is linear with time for 40 to 50 min and causes a six- to eight-fold increase over control levels (10 to 15 picomole cAMP/mg cellular protein) at steady state. cAMP and agents that increase cAMP production inhibit Cl--independent Na+ influx into the isolated enterocytes whereas chlorpromazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na+ entry. Correlation between cellular cAMP levels and the magnitude of Na+ influx provides evidence for a cAMP-mediated control of intestinal Na+ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na+ during induction of intestinal secretion. The effect of cAMP on Na+ but not Cl- influx preparations can be partially explained in terms of a cAMP-regulated Na+/H+ neutral exchange system. Data on the coupling relationship between Na+ transport and the intra- and extracellular pH in the enterocytes show that an amiloride-sensitive electroneutral Na+/H+ exchange process occurs. This coupling between Na+ and H+ is partially inhibited by CT and dbcAMP, suggesting that the Na+/H+ exchange may be a cAMP-regulated process. 31 references, 32 figures, 5 tables

  3. Action of cholera toxin in the intestinal epithelial cells

    International Nuclear Information System (INIS)

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with the cell membrane. This involves a large number (17 million per cell) of high affinity binding sites which belong to a single class. Binding of biologically active 125I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected in the isolated cells. The response (elevation of cellular cAMP) of the enterocytes to cholera toxin is linear with time for 40-50 min and causes a six- to eight-fold increase over control levels at steady stae. cAMP and agents that increase cAMP production inhibit Cl--independent Na+ influx into the isolated enterocytes whereas chlorporomazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na+ entry. Correlation between cellular cAMP levels and the magnitude of Na+ influx into the enterocytes provides evidence for a cAMP-mediated control of intestinal Na+ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT and Na+ during induction of intestinal secretion. The effect of cAMP on Na+ but no Cl- influx in our villus cell preparation can be partially explained in terms of a cAMP-regulated Na+/H+ neutral exchange system

  4. Mechanism of Interferon-γ–Induced Increase in T84 Intestinal Epithelial Tight Junction

    OpenAIRE

    Boivin, Michel A.; Roy, Praveen K.; Bradley, Angela; Kennedy, John C.; Rihani, Tuhama; Ma, Thomas Y.

    2009-01-01

    Interferon-γ (IFN-γ) is an important proinflammatory cytokine that plays a central role in the intestinal inflammatory process of inflammatory bowel disease. IFN-γ induced disturbance of the intestinal epithelial tight junction (TJ) barrier has been postulated to be an important mechanism contributing to intestinal inflammation. The intracellular mechanisms that mediate the IFN-γ induced increase in intestinal TJ permeability remain unclear. The aim of this study was to examine the role of th...

  5. Differentiation-dependent activation of the human intestinal alkaline phosphatase promoter by HNF-4 in intestinal cells

    DEFF Research Database (Denmark)

    Olsen, Line; Bressendorff, Simon; Troelsen, Jesper T;

    2005-01-01

    The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the...... of HNF-4alpha to stimulate the expression from the ALPI promoter was investigated in the nonintestinal Hela cell line. Cotransfection with an HNF-4alpha expression vector demonstrated a direct activation of the ALPI promoter through this -94 to -82 element. EMSA showed that HNF-4alpha from nuclear...... extracts of differentiated intestinal epithelial cells (Caco-2) bound with high affinity to the predicted HNF-4 binding site. A 521 bp promoter fragment containing the HNF-4 binding site demonstrated a differentiation-dependent increase in promoter activity in Caco-2 cells. The presence of the HNF-4...

  6. Kruppel-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

  7. Dual Effects Exerted in Vitro by Micromolar Concentrations of Deoxynivalenol on Undifferentiated Caco-2 Cells

    Science.gov (United States)

    Manda, Gina; Mocanu, Mihaela Andreea; Marin, Daniela Eliza; Taranu, Ionelia

    2015-01-01

    Contamination of crops used for food and feed production with Fusarium mycotoxins, such as deoxynivalenol (DON), raise important health and economic issues all along the food chain. Acute exposure to high DON concentrations can alter the intestinal barrier, while chronic exposure to lower doses may exert more subtle effects on signal transduction pathways, leading to disturbances in cellular homeostasis. Using real-time cellular impedance measurements, we studied the effects exerted in vitro by low concentrations of DON (0.37–1.50 μM), relevant for mycotoxin-contaminated food, on the proliferation of undifferentiated Caco-2 cells presenting a tumorigenic phenotype. A 1.5 μM concentration of DON maintained cell adherence of non-proliferating Caco-2 cells, whilst arresting the growth of actively proliferating cells compared with control Caco-2 cells in vitro. At 0.37 μM, DON enhanced Caco-2 cell metabolism, thereby triggering a moderate increase in cell proliferation. The results of the current study suggested that low concentrations of DON commonly detected in food may either limit or sustain the proliferation of colon cancer cells, depending on their proliferation status and on DON concentration. Soluble factors released by Lactobacillus strains can partially counteract the inhibitory action of DON on actively proliferating colon cancer cells. The study also emphasized that real-time cellular impedance measurements were a valuable tool for investigating the dynamics of cellular responses to xenobiotics. PMID:25690693

  8. 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).

  9. 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

  10. Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

    OpenAIRE

    Koch, Stefan; Capaldo, Christopher T.; Samarin, Stanislav; Nava, Porfirio; Neumaier, Irmgard; Skerra, Arne; Sacks, David B; Parkos, Charles A.; Nusrat, Asma

    2009-01-01

    Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating c...

  11. Interferon-γ regulates intestinal epithelial homeostasis through converging β-catenin signaling pathways

    OpenAIRE

    Nava, Porfirio; Koch, Stefan; Laukoetter, Mike G.; Lee, Winston Y.; Kolegraff, Keli; Capaldo, Christopher T.; Beeman, Neal; Addis, Caroline; Gerner-Smidt, Kirsten; Neumaier, Irmgard; Skerra, Arne; Li, Linheng; Parkos, Charles A.; Nusrat, Asma

    2010-01-01

    Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-γ (IFN-γ) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-β-catenin and Wingless-Int (Wnt)-β-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-γ resulted in activation of β-catenin through AKT, followed by induction of the secreted Wnt inhibitor...

  12. 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.

  13. 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

    2010-12-01

    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. PMID:21070005

  14. 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. PMID:26830743

  15. 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 ga

  16. 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.

  17. Titanium Dioxide Particle Type and Concentration Influence the Inflammatory Response in Caco-2 Cells.

    Science.gov (United States)

    Tada-Oikawa, Saeko; Ichihara, Gaku; Fukatsu, Hitomi; Shimanuki, Yuka; Tanaka, Natsuki; Watanabe, Eri; Suzuki, Yuka; Murakami, Masahiko; Izuoka, Kiyora; Chang, Jie; Wu, Wenting; Yamada, Yoshiji; Ichihara, Sahoko

    2016-01-01

    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. PMID:27092499

  18. Titanium Dioxide Particle Type and Concentration Influence the Inflammatory Response in Caco-2 Cells

    Science.gov (United States)

    Tada-Oikawa, Saeko; Ichihara, Gaku; Fukatsu, Hitomi; Shimanuki, Yuka; Tanaka, Natsuki; Watanabe, Eri; Suzuki, Yuka; Murakami, Masahiko; Izuoka, Kiyora; Chang, Jie; Wu, Wenting; Yamada, Yoshiji; Ichihara, Sahoko

    2016-01-01

    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. PMID:27092499

  19. Monitoring in real time the cytotoxic effect of Clostridium difficile upon the intestinal epithelial cell line HT29.

    Science.gov (United States)

    Valdés, Lorena; Gueimonde, Miguel; Ruas-Madiedo, Patricia

    2015-12-01

    The incidence and severity of Clostridium difficile infections (CDI) has been increased not only among hospitalized patients, but also in healthy individuals traditionally considered as low risk population. Current treatment of CDI involves the use of antibiotics to eliminate the pathogen, although recurrent relapses have also been reported. For this reason, the search of new antimicrobials is a very active area of research. The strategy to use inhibitors of toxin's activity has however been less explored in spite of being a promising option. In this regard, the lack of fast and reliable in vitro screening methods to search for novel anti-toxin drugs has hampered this approach. The aim of the current study was to develop a method to monitor in real time the cytotoxicity of C. difficile upon the human colonocyte-like HT29 line, since epithelial intestinal cells are the primary targets of the toxins. The label-free, impedance based RCTA (real time cell analyser) technology was used to follow overtime the behaviour of HT29 in response to C. difficile LMG21717 producing both A and B toxins. Results obtained showed that the selection of the medium to grow the pathogen had a great influence in obtaining toxigenic supernatants, given that some culture media avoided the release of the toxins. A cytotoxic dose- and time-dependent effect of the supernatant obtained from GAM medium upon HT29 and Caco2 cells was detected. The sigmoid-curve fit of data obtained with HT29 allowed the calculation of different toxicological parameters, such as EC50 and LOAEL values. Finally, the modification in the behaviour of HT29 reordered in the RTCA was correlated with the cell rounding effect, typically induced by these toxins, visualized by time-lapsed captures using an optical microscope. Therefore, this RTCA method developed to test cytotoxicity kinetics of C. difficile supernatants upon IEC could be a valuable in vitro model for the screening of new anti-CDI agents. PMID:26436983

  20. Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

    OpenAIRE

    Lebeer, S.; Claes, I.J.; Tytgat, H.L.P.; Verhoeven, T.L.A.; Marien, E.; Ossowski, von, I.; Reunanen, J.; Palva, A.; Vos, de, W.M.; Keersmaecker, de, S.C.; Vanderleyden, J.

    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 epithelia...

  1. Comparative evaluation of nano-CuO crossing Caco-2 cell monolayers and cellular uptake

    International Nuclear Information System (INIS)

    Different concentrations of CuSO4, 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 (Papp) of CuSO4 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 Papp 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 Papp value of Caco-2 cells incubated with nano-CuO was significantly higher than that obtained with CuSO4. 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 CuSO4 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 CuSO4 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

  2. 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.

  3. Reciprocal regulation of the primary sodium absorptive pathways in rat intestinal epithelial cells

    OpenAIRE

    Coon, Steven; Kekuda, Ramesh; Saha, Prosenjit; Sundaram, Uma

    2010-01-01

    Sodium absorption in the mammalian small intestine occurs predominantly by two primary pathways that include Na/H exchange (NHE3) and Na-glucose cotransport (SGLT1) on the brush border membrane (BBM) of villus cells. However, whether NHE3 and SGLT1 function together to regulate intestinal sodium absorption is unknown. Nontransformed small intestinal epithelial cells (IEC-18) were transfected with either NHE3 or SGLT1 small interfering RNAs (siRNAs) and were grown in confluent monolayers on tr...

  4. 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 by a c...

  5. Estimation of digestive stability and bioavailability of chlorophylls by an in vitro digestion/Caco-2 cell culture model

    OpenAIRE

    Gallardo Guerrero, Lourdes; Gandul-Rojas, Beatriz; Mínguez Mosquera, María Isabel

    2006-01-01

    The aim of this work was to study the effect of food matrix both on the chlorophyll pigment transformations and on their micellarization as a measure of bioavailability, using an two-stage in vitro digestion model. We also validate the above mentioned bioavailability measurement by evaluating the absorption of the micellarized chlorophyll pigments by Caco-2 human intestinal cell cultures.

  6. Apomorphine and its esters: Differences in Caco-2 cell permeability and chylomicron affinity.

    Science.gov (United States)

    Borkar, Nrupa; Chen, Zhizhong; Saaby, Lasse; Müllertz, Anette; Håkansson, Anders E; Schönbeck, Christian; Yang, Mingshi; Holm, René; Mu, Huiling

    2016-07-25

    Oral delivery of apomorphine via prodrug principle may be a potential treatment for Parkinson's disease. The purpose of this study was to investigate the transport and stability of apomorphine and its esters across Caco-2 cell monolayer and their affinity towards chylomicrons. Apomorphine, monolauroyl apomorphine (MLA) and dilauroyl apomorphine (DLA) were subjected to apical to basolateral (A-B) and basolateral to apical (B-A) transport across Caco-2 cell monolayer. The stability of these compounds was also assessed by incubation at intestinal pH and physiological pH with and without Caco-2 cells. Molecular dynamics (MD) simulations were performed to understand the stability of the esters on a molecular level. The affinity of the compounds towards plasma derived chylomicrons was assessed. The A-B transport of intact DLA was about 150 times lower than the transport of apomorphine. In contrast, MLA was highly unstable in the aqueous media leading to apomorphine appearance basolaterally. MD simulations possibly explained the differences in hydrolysis susceptibilities of DLA and MLA. The affinity of apomorphine diesters towards plasma derived chylomicrons provided an understanding of their potential lymphatic transport. The intact DLA transport is not favorable; therefore, the conversion of DLA to MLA is an important step for intestinal apomorphine absorption. PMID:27282537

  7. 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.

  8. IL-1β stimulation of CCD-18co myofibroblasts enhances repair of epithelial monolayers through Wnt-5a.

    Science.gov (United States)

    Raymond, Meera; Marchbank, Tania; Moyer, Mary P; Playford, Raymond J; Sanderson, Ian R; Kruidenier, Laurens

    2012-12-01

    Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/β-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1β-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1β-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1β-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1β stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1β-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1β-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound

  9. 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. PMID:25653232

  10. Bacterial Wall Components such as Lipothecoid Acid, Peptidoglycan, Liposaccharide and Lipid A Stimulate Cell Proliferation in Intestinal Epithelial Cells

    OpenAIRE

    Olaya, Jaime H.; Neopikhanov, Vadim; Söderman, Charlotte; Uribe, Andrés

    2011-01-01

    Earlier studies indicate that the microflora contains mitogens to intestinal epithelial cells. Our aim is to examine whether cell wall components of both Gram-negative and positive bacteria influence cell proliferation in small intestinal and colonic epithelial cells. A human colonic epithelial cell line from adenocarcinoma (IEC-6) and a nontransformed small intestinal cell line from germ-free rats (LS-123) were incubated with (a) lipothecoid acid from Streptococcus faecalis at 1.56–50 ...

  11. Dietary whole-grain wheat increases intestinal levels of bifidobacteria in humans and bifidobacterial abundance is negatively correlated with the effect of fecal water on trans-epithelial resistance in vitro

    DEFF Research Database (Denmark)

    Christensen, Ellen Gerd; Licht, Tine Rask; Kristensen, M.;

    composition in post-menopausal women following a 12-week energy restricted intervention with whole-grain wheat (WW, n=37) or refined wheat (RW, n=33). The WW intervention significantly increased the relative abundance of Bifidobacterium. Caco-2 cells were exposed to fecal water to determine effects of the......Consumption of whole grain products are considered to have beneficial effects on human health including decreased risk of cardiovascular disease. However, effects on gut microbial composition have only been studied limitedly. We used quantitative PCR to determine changes in the gut bacterial...... bacterial community metabolites on the trans-epithelial resistance (TER). Fecal water increased TER independent of diet, indicating that commensal bacteria provide metabolites facilitating an increase in intestinal integrity. TER was unexpectedly found to be negatively correlated to the relative abundance...

  12. 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

    2013-03-01

    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

  13. 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.

    2013-01-01

    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-gp inhibitor, verapamil significantly

  14. Commensal Bacteria and Epithelial Cross Talk in the Developing Intestine

    OpenAIRE

    Rautava, Samuli; Walker, W. Allan

    2007-01-01

    Indigenous intestinal microbes have co-evolved with the intestinal immune system to form a symbiotic ecosystem. In the postnatal period, intestinal microbes provide the developing gut with stimuli that are necessary for healthy maturation of the intestinal immune system. Cross talk between the host and commensal microbes is an essential component of gut homeostasis mechanisms also in later life. During recent years, innovative research has shed light on the molecular mechanisms of these inter...

  15. Neutrophil Interactions with Epithelial Expressed ICAM-1 Enhances Intestinal Mucosal Wound Healing

    Science.gov (United States)

    Sumagin, R; Brazil, JC; Nava, P; Nishio, H; Alam, A; Luissint, AC; Weber, DA; Neish, AS; Nusrat, A; Parkos, CA

    2015-01-01

    A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. While epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 plays an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing. PMID:26732677

  16. Lactobacillus acidophilus stimulates intestinal P-glycoprotein expression via a c-Fos/c-Jun-dependent mechanism in intestinal epithelial cells.

    Science.gov (United States)

    Priyamvada, Shubha; Anbazhagan, Arivarasu N; Kumar, Anoop; Soni, Vikas; Alrefai, Waddah A; Gill, Ravinder K; Dudeja, Pradeep K; Saksena, Seema

    2016-04-15

    Our previous studies showed that Lactobacillus acidophilus (LA) culture supernatant (CS) increased P-glycoprotein [Pgp/multidrug resistance 1 (MDR1)] function, expression, and promoter activity in Caco-2 cells. The current studies were designed to elucidate the molecular mechanisms mediating the stimulatory effects of LA CS on Pgp promoter activity. Deletion analysis indicated that the LA CS response element(s) is located in the -172/+428-bp region, and sequence analysis of this region revealed three potential binding sites for c-Fos or c-Jun: proximal activating protein (AP) 1a (-119/-98 bp), distal AP1b (-99/-78 bp), and AP1c (+175/+196 bp). LA CS (24 h) showed an approximately twofold increase in the protein expression of c-Fos and c-Jun in Caco-2 cells. Electrophoretic mobility shift assay showed that LA CS markedly increased the binding of Caco-2 nuclear proteins to AP1a and AP1b, but not AP1c. The DNA-protein complex was completely eliminated by c-Fos antibody, while c-Jun antibody partially eliminated the complex. Chromatin immunoprecipitation analysis also showed that LA CS enhanced the association of c-Fos and c-Jun (by ∼4- and 1.5-fold, respectively) with endogenous Pgp promoter in Caco-2 cells (p-172/+1). Interestingly, overexpression of c-Fos or c-Jun activated Pgp promoter by nearly twofold each. This increase was further enhanced (∼14-fold) when c-Fos and c-Jun were simultaneously overexpressed, suggesting that the presence of one of these transcription factors potentiates the effect of the other. These studies, for the first time, provide evidence for the involvement of c-Fos/c-Jun in stimulation of Pgp gene expression by LA CS in the human intestine. PMID:26867563

  17. 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...

  18. A Comparative Study on Rat Intestinal Epithelial Cells and Resident Gut Bacteria (ii) Effect of Arsenite

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In order to use facultative gut bacteria as an alternate to animals for the initial gastrointestinal toxicity screening of heavy metals, a comparative study on rat intestinal epithelial cells and resident gut bacteria was undertaken.Methods in vitro growth rate of four gut bacteria, dehydrogenase (DHA) and esterase (EA) activity test, intestinal epithelial and bacterial cell membrane enzymes and in situ effect of arsenite were analysed. Results Growth profile of mixed resident population of gut bacteria and pure isolates of Escherichia coli, Pseudomonas sp., Lactobacillus sp., and Staphylococcus sp.revealed an arsenite (2-20 ppm) concentration-dependent inhibition. The viability pattern of epithelial cells also showed similar changes. DHA and EA tests revealed significant inhibition (40%-72%) with arsenite exposure of 5 and 10 ppm in isolated gut bacteria and epithelial cells. Decrease in membrane alkaline phosphatase and Ca2+-Mg2+-ATPase activities was in the range of 33%-55% in four bacteria at the arsenite exposure of 10 ppm, whereas it was 60%-65% in intestinal epithelial villus cells. in situ incubation of arsenite using intestinal loops also showed more or less similar changes in membrane enzymes of resident gut bacterial population and epithelial cells. Conclusion The results indicate that facultative gut bacteria can be used as suitable in vitro model for the preliminary screening of arsenical gastrointestinal cytotoxic effects.

  19. Effects of propofol on damage of rat intestinal epithelial cells induced by heat stress and lipopolysaccharides

    Directory of Open Access Journals (Sweden)

    J. Tang

    2013-06-01

    Full Text Available 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.

  20. Effects of propofol on damage of rat intestinal epithelial cells induced by heat stress and lipopolysaccharides

    International Nuclear Information System (INIS)

    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

  1. 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.

  2. Cysteine protease activity of feline Tritrichomonas foetus promotes adhesion-dependent cytotoxicity to intestinal epithelial cells.

    Science.gov (United States)

    Tolbert, M K; Stauffer, S H; Brand, M D; Gookin, J L

    2014-07-01

    Trichomonads are obligate protozoan parasites most renowned as venereal pathogens of the reproductive tract of humans and cattle. Recently, a trichomonad highly similar to bovine venereal Tritrichomonas foetus but having a unique tropism for the intestinal tract was recognized as a significant cause of colitis in domestic cats. Despite a high prevalence, worldwide distribution, and lack of consistently effective drugs for treatment of the infection, the cellular mechanisms of T. foetus pathogenicity in the intestinal tract have not been examined. The aims of this study were to determine the pathogenic effect of feline T. foetus on porcine intestinal epithelial cells, the dependence of T. foetus pathogenicity on adhesion of T. foetus to the intestinal epithelium, and the identity of mediators responsible for these effects. Using an in vitro coculture approach to model feline T. foetus infection of the intestinal epithelium, these studies demonstrate that T. foetus promotes a direct contact-dependent activation of intestinal epithelial cell apoptosis signaling and progressive monolayer destruction. Moreover, these pathological effects were demonstrated to be largely dependent on T. foetus cell-associated cysteine protease activity. Finally, T. foetus cysteine proteases were identified as enabling cytopathic effects by promoting adhesion of T. foetus to the intestinal epithelium. The present studies are the first to examine the cellular mechanisms of pathogenicity of T. foetus toward the intestinal epithelium and support further investigation of the cysteine proteases as virulence factors in vivo and as potential therapeutic targets for ameliorating the pathological effects of intestinal trichomonosis. PMID:24752513

  3. In vitro toxicity of different-sized ZnO nanoparticles in Caco-2 cells

    Science.gov (United States)

    Kang, Tianshu; Guan, Rongfa; Chen, Xiaoqiang; Song, Yijuan; Jiang, Han; Zhao, Jin

    2013-11-01

    There has been rapid growth in nanotechnology in both the public and private sectors worldwide, but concern about nanosafety exists. To assess size-dependent cytotoxicity on human cancer cells, we studied the cytotoxic effect of three kinds of zinc oxide nanoparticles (ZnO NPs) on human epithelial colorectal adenocarcinoma (Caco-2) cells. Nanoparticles were first characterized by size, distribution, and intensity. Multiple assays have been adopted to measure the cell activity and oxidative stress. The cytotoxicity of ZnO NPs was time dependent and dose dependent. The 24-h exposure was chosen to confirm the viability and accessibility of the cells and taken as the appropriate time for the following test system. The IC50 value was found at a low concentration. The oxidative stress elicited a significant reduction in glutathione with increase in reactive oxygen species and lactate dehydrogenase. The toxicity resulted in a deletion of cells in the G1 phase and an accumulation of cells in the S and G2/M phases. One type of metallic oxide (ZnO) exerted different cytotoxic effects according to different particle sizes. Data from the previous experiments showed that 26-nm ZnO NPs appeared to have the highest toxicity to Caco-2 cells. The study demonstrated the toxicity of ZnO NPs to Caco-2 cells and the impact of particle size, which could be useful in the medical applications.

  4. Na+-independent phosphate transport in Caco2BBE cells

    Science.gov (United States)

    Candeal, Eduardo; Caldas, Yupanqui A.; Guillén, Natalia; Levi, Moshe

    2014-01-01

    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, SO42−, 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. PMID:25298422

  5. 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.

  6. 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.

  7. Permeability of rhynchophylline across human intestinal cell in vitro.

    Science.gov (United States)

    Ma, Bo; Wang, Jing; Sun, Jing; Li, Ming; Xu, Huibo; Sun, Guibo; Sun, Xiaobo

    2014-01-01

    Rhynchophylline (Rhy) is the major component of Uncaria species, which is used in Chinese traditional medicine for the treatment of central nervous system disorders. However, its oral bioavailability has not been known. This study aims to investigate the intestinal permeability and related mechanisms of Rhy using cultured human epithelial Caco-2 cells. The cytotoxicity of Rhy on Caco-2 cells was evaluated with MTT assay. The effect of Rhy on the integrity of Caco-2 cell monolayer was assayed with transepithelial electrical resistance. The permeability of Rhy across cell monolayer was assayed by measuring Rhy quantity in received side with HPLC. The effect of Rhy on the expression of P-glycoprotein and MDR1 was detected with Western blot and flow cytometry, respectively. In the concentration of Rhy, which did not produce toxicity on cell viability and integrity of Caco-2 cell monolayer, Rhy crossed the monolayer with velocity 2.76~5.57×10^-6 cm/sec and 10.68~15.66×10^-6 cm/sec from apical to basolateral side and from basolateral to apical side, respectively. The permeability of Rhy was increased by verapamil, a P-glycoprotein inhibitor, or rhodamine123, a P-glycoprotein substrate. Rhy revealed an induction effect on P-glycoprotein expression in Caco-2 cells. These results demonstrate the low permeability of Rhy in intro, and suggest that P-glycoprotein may underlie the mechanism. PMID:24966905

  8. Oral Treatment with Extract of Agaricus blazei Murill Enhanced Th1 Response through Intestinal Epithelial Cells and Suppressed OVA-Sensitized Allergy in Mice

    Directory of Open Access Journals (Sweden)

    Go Bouike

    2011-01-01

    Full Text Available To clarify the mechanism of the antiallergic activity of Agaricus blazei Murill extract (ABME, the present paper used an in vivo allergy model and an in vitro intestinal gut model. During OVA sensitization, the serum IgE levels decreased significantly in ABME group. Interleukin (IL-4 and -5 produced from OVA-restimulated splenocytes was significantly decreased, and anti-CD3ε/CD28 antibody treatment also reduced IL-10, -4, and -5 production and increased IFN-γ production in ABME group. These results suggest that oral administration of ABME improves Th1/Th2 balance. Moreover, a coculture system constructed of Caco-2 cells and splenocytes from OT-II mice or RAW 264.7 cells indicated that the significant increases in IFN-γ production by ABME treatment. Therefore, it was concluded that the antiallergic activity of ABME was due to the activation of macrophages by epithelial cells and the promotion of the differentiation of naïve T cells into Th1 cells in the immune.

  9. Circadian regulation of epithelial functions in the intestine

    Czech Academy of Sciences Publication Activity Database

    Pácha, Jiří; Sumová, Alena

    2013-01-01

    Roč. 208, č. 1 (2013), s. 11-24. ISSN 1748-1708 R&D Projects: GA ČR(CZ) GAP303/10/0969; GA ČR(CZ) GAP303/11/0668 Institutional support: RVO:67985823 Keywords : circadian rhythms * intestine * colon * proliferation * digestion * intestinal transport Subject RIV: ED - Physiology Impact factor: 4.251, year: 2013

  10. Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis by intestinal epithelial cells.

    Science.gov (United States)

    Pott, Johanna; Basler, Tina; Duerr, Claudia U; Rohde, Manfred; Goethe, Ralph; Hornef, Mathias W

    2009-12-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal-oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-kappaB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection. PMID:19681906

  11. E. coli Nissle 1917 Affects Salmonella Adhesion to Porcine Intestinal Epithelial Cells

    OpenAIRE

    Schierack, P.; Kleta, S; Tedin, K.; Babila, J.T.; Oswald, S.; Oelschlaeger, T A; Hiemann, R.; Paetzold, S; Wieler, L H

    2011-01-01

    Background The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. Methodology/Principal Findings In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including in...

  12. Vectorial transport of fexofenadine across Caco-2 cells: involvement of apical uptake and basolateral efflux transporters.

    Science.gov (United States)

    Ming, Xin; Knight, Beverly M; Thakker, Dhiren R

    2011-10-01

    Fexofenadine is a nonsedative antihistamine that exhibits good oral bioavailability despite its zwitterionic chemical structure and efflux by P-gp. Evidence exists that multiple uptake and efflux transporters play a role in hepatic disposition of fexofenadine. However, the roles of specific transporters and their interrelationship in intestinal absorption of this drug are unclear. This study was designed to elucidate vectorial absorptive transport of fexofenadine across Caco-2 cells involving specific apical uptake and efflux transporters as well as basolateral efflux transporters. Studies with cellular models expressing single transporters showed that OATP2B1 expression stimulated uptake of fexofenadine at pH 6.0. Apical uptake of fexofenadine into Caco-2 cells was decreased by 45% by pretreatment with estrone 3-sulfate, an OATP inhibitor, at pH 6.0 but not at pH 7.4, indicating that OATP2B1 mediates apical uptake of fexofenadine into these cells. Examination of fexofenadine efflux from preloaded Caco-2 cells in the presence or absence of (i) the MRP inhibitor MK-571 and (ii) the P-gp inhibitor GW918 showed that apical efflux is predominantly mediated by P-gp, with a small contribution by MRP2, whereas basolateral efflux is predominantly mediated by MRP3. These results also showed that while OSTαβ is functionally active in the basolateral membrane of Caco-2 cells, it does not play a role in the export of fexofenadine. MK-571 decreased the absorptive transport of fexofenadine by 17%. However, the decrease in absorptive transport by MK-571 was 42% when P-gp was inhibited by GW918. The results provide a novel insight into a vectorial transport system mainly consisting of apical OATP2B1 and basolateral MRP3 that may play an important role in delivering hydrophilic anionic and zwitterionic drugs such as pravastatin and fexofenadine into systemic circulation upon oral administration. PMID:21780830

  13. Metabolites produced by probiotic Lactobacilli rapidly increase glucose uptake by Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Buddington Randal K

    2010-01-01

    Full Text Available Abstract Background Although probiotic bacteria and their metabolites alter enterocyte gene expression, rapid, non-genomic responses have not been examined. The present study measured accumulation of tracer (2 μM glucose by Caco-2 cells after exposure for 10 min or less to a chemically defined medium (CDM with different monosaccharides before and after anaerobic culture of probiotic Lactobacilli. Results Growth of L. acidophilus was supported by CDM with 110 mM glucose, fructose, and mannose, but not with arabinose, ribose, and xylose or the sugar-free CDM. Glucose accumulation was reduced when Caco-2 cells were exposed for 10 min to sterile CDM with glucose (by 92%, mannose (by 90%, fructose (by 55%, and ribose (by 16%, but not with arabinose and xylose. Exposure of Caco-2 cells for 10 min to bacteria-free supernatants prepared after exponential (48 h and stationary (72 h growth phases of L. acidophilus cultured in CDM with 110 mM fructose increased glucose accumulation by 83% and 45%, respectively; exposure to a suspension of the bacteria had no effect. The increase in glucose accumulation was diminished by heat-denaturing the supernatant, indicating the response of Caco-2 cells is triggered by as yet unknown heat labile bacterial metabolites, not by a reduction in CDM components that decrease glucose uptake. Supernatants prepared after anaerobic culture of L. gasseri, L. amylovorus, L. gallinarum, and L. johnsonii in the CDM with fructose increased glucose accumulation by 83%, 32%, 27%, and 14%, respectively. Conclusion The rapid, non-genomic upregulation of SGLT1 by bacterial metabolites is a heretofore unrecognized interaction between probiotics and the intestinal epithelium.

  14. Impact Assessment of Cadmium Toxicity and Its Bioavailability in Human Cell Lines (Caco-2 and HL-7702

    Directory of Open Access Journals (Sweden)

    Rukhsanda Aziz

    2014-01-01

    Full Text Available Cadmium (Cd is a widespread environmental toxic contaminant, which causes serious health-related problems. In this study, human intestinal cell line (Caco-2 cells and normal human liver cell line (HL-7702 cells were used to investigate the toxicity and bioavailability of Cd to both cell lines and to validate these cell lines as in vitro models for studying Cd accumulation and toxicity in human intestine and liver. Results showed that Cd uptake by both cell lines increased in a dose-dependent manner and its uptake by Caco-2 cells (720.15 µg mg−1 cell protein was significantly higher than HL-7702 cells (229.01 µg mg−1 cell protein at 10 mg L−1. A time- and dose-dependent effect of Cd on cytotoxicity assays (LDH release, MTT assay was observed in both Cd-treated cell lines. The activities of antioxidant enzymes and differentiation markers (SOD, GPX, and AKP of the HL-7702 cells were higher than those of Caco-2 cells, although both of them decreased significantly with raising Cd levels. The results from the present study indicate that Cd above a certain level inhibits cellular antioxidant activities and HL-7702 cells are more sensitive to Cd exposure than Caco-2 cells. However, Cd concentrations <0.5 mg L−1 pose no toxic effects on both cell lines.

  15. 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.

  16. 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

    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 of...

  17. Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches

    DEFF Research Database (Denmark)

    Giannakis, Marios; Stappenbeck, Thaddeus S; Mills, Jason C;

    2006-01-01

    We have sequenced 36,641 expressed sequence tags from laser capture microdissected adult mouse gastric and small intestinal epithelial progenitors, obtaining 4031 and 3324 unique transcripts, respectively. Using Gene Ontology (GO) terms, each data set was compared with cDNA libraries from intact ...

  18. 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 epithelium, however

  19. Epithelial structure and function in the hen lower intestine

    DEFF Research Database (Denmark)

    Laverty, G.; Elbrønd, Vibeke Sødring; Árnason, Sigvatur S.;

    2006-01-01

    and the proportion of 'mitrochondrial-rich' (MR) cells. The latter may be the sites of proton secretion in the lower intestine. A cAMP-activated chloride secretion pathway is also present in both colon and coprodeum, and may be mediated by a CFTR-like Cl- channel. There are still a number of...

  20. Mechanism of Interferon-γ–Induced Increase in T84 Intestinal Epithelial Tight Junction

    Science.gov (United States)

    Boivin, Michel A.; Roy, Praveen K.; Bradley, Angela; Kennedy, John C.; Rihani, Tuhama

    2009-01-01

    Interferon-γ (IFN-γ) is an important proinflammatory cytokine that plays a central role in the intestinal inflammatory process of inflammatory bowel disease. IFN-γ induced disturbance of the intestinal epithelial tight junction (TJ) barrier has been postulated to be an important mechanism contributing to intestinal inflammation. The intracellular mechanisms that mediate the IFN-γ induced increase in intestinal TJ permeability remain unclear. The aim of this study was to examine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in the regulation of the IFN-γ induced increase in intestinal TJ permeability using the T84 intestinal epithelial cell line. IFN-γ caused an increase in T84 intestinal epithelial TJ permeability and depletion of TJ protein, occludin. The IFN-γ induced increase in TJ permeability and alteration in occludin protein was associated with rapid activation of PI3-K; and inhibition of PI3-K activation prevented the IFN-γ induced effects. IFN-γ also caused a delayed but more prolonged activation of nuclear factor-κB (NF-κB); inhibition of NF-κB also prevented the increase in T84 TJ permeability and alteration in occludin expression. The IFN-γ induced activation of NF-κB was mediated by a cross-talk with PI3-K pathway. In conclusion, the IFN-γ induced increase in T84 TJ permeability and alteration in occludin protein expression were mediated by the PI3-K pathway. These results show for the first time that the IFN-γ modulation of TJ protein and TJ barrier function is regulated by a cross-talk between PI3-K and NF-κB pathways. PMID:19128033

  1. Mechanism of interferon-gamma-induced increase in T84 intestinal epithelial tight junction.

    Science.gov (United States)

    Boivin, Michel A; Roy, Praveen K; Bradley, Angela; Kennedy, John C; Rihani, Tuhama; Ma, Thomas Y

    2009-01-01

    Interferon-gamma (IFN-gamma) is an important proinflammatory cytokine that plays a central role in the intestinal inflammatory process of inflammatory bowel disease. IFN-gamma induced disturbance of the intestinal epithelial tight junction (TJ) barrier has been postulated to be an important mechanism contributing to intestinal inflammation. The intracellular mechanisms that mediate the IFN-gamma induced increase in intestinal TJ permeability remain unclear. The aim of this study was to examine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in the regulation of the IFN-gamma induced increase in intestinal TJ permeability using the T84 intestinal epithelial cell line. IFN-gamma caused an increase in T84 intestinal epithelial TJ permeability and depletion of TJ protein, occludin. The IFN-gamma induced increase in TJ permeability and alteration in occludin protein was associated with rapid activation of PI3-K; and inhibition of PI3-K activation prevented the IFN-gamma induced effects. IFN-gamma also caused a delayed but more prolonged activation of nuclear factor-kappaB (NF-kappaB); inhibition of NF-kappaB also prevented the increase in T84 TJ permeability and alteration in occludin expression. The IFN-gamma induced activation of NF-kappaB was mediated by a cross-talk with PI3-K pathway. In conclusion, the IFN-gamma induced increase in T84 TJ permeability and alteration in occludin protein expression were mediated by the PI3-K pathway. These results show for the first time that the IFN-gamma modulation of TJ protein and TJ barrier function is regulated by a cross-talk between PI3-K and NF-kappaB pathways. PMID:19128033

  2. Alisertib Induces Cell Cycle Arrest, Apoptosis, Autophagy and Suppresses EMT in HT29 and Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Bao-Jun Ren

    2015-12-01

    Full Text Available 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.

  3. 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. PMID:27153755

  4. 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...

  5. Modulation of human enteric epithelial barrier and ion transport function by Peyer's patch lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Jie Chen; Lai-Ling Tsang; Lok-Sze Ho; Dewi K.Rowlands; Jie-Ying Gao; Chuen-Pei Ng; Yiu-Wa Chung; Hsiao-Chang Chan

    2004-01-01

    AIM: To investigate the role of Peyer′s patch lymphocytes in the regulation of enteric epithelial barrier and ion transport function in homeostasis and host defense.METHODS: Mouse Peyer′s patch lymphocytes were cocultured with human intestinal epithelial cell line Caco-2either in the mixed or separated (isolated but permeable compartments) culture configuration. Barrier and transport functions of the Caco-2 epithelial monolayers were measured with short-circuit current (ISC) technique. Release of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and cytokine mRNA expression was analyzed by semi-quantitative RT-PCR. Barrier and iontransport functions of both culture conditions following exposure to Shigella lipopolysaccharide (LPS) were also examined.RESULTS: The transepithelial resistance (TER) of the epithelial monolayers co-cultured with Peyer′s patch lymphocytes was maintained whereas that of the Caco-2 monolayers alone significantly decreased after eight days in culture.The forskolin-induced anion secretion, in either absence or presence of LPS, was significantly suppressed in the both co-cultures as compared with the Caco-2 cells alone.Furthermore, only the mixed co-culture condition induced the expression and release of mIL-6 from Peyer′s patch lymphocytes, which could be further enhanced by LPS.However, both co-culture conditions suppressed expression and release of epithelial hIL-8 under the unstimulated conditions, while the treatment with LPS stimulated their hIL-8 expression and release.CONCLUSION: Peyer′s patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact and cytokine signaling.

  6. 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

  7. 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.

  8. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    International Nuclear Information System (INIS)

    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

  9. 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.

  10. Ussing Chamber Technique to Measure Intestinal Epithelial Permeability.

    Science.gov (United States)

    Vidyasagar, Sadasivan; MacGregor, Gordon

    2016-01-01

    Epithelial cells are polarized and have tight junctions that contribute to barrier function. Assessment of barrier function typically involves measurement of electrophysiological parameters or movement of nonionic particles across an epithelium. Here, we describe measurement of transepithelial electrical conductance or resistance, determination of dilution potential, and assessment of flux of nonionic particles such as dextran or mannitol, with particular emphasis on Ussing chamber techniques. PMID:27246022

  11. Interferon-γ suppresses intestinal epithelial aquaporin-1 expression via Janus kinase and STAT3 activation.

    Directory of Open Access Journals (Sweden)

    Michael S Dicay

    Full Text Available Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1 as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway.

  12. Interferon-γ regulates intestinal epithelial homeostasis through converging β-catenin signaling pathways

    Science.gov (United States)

    Nava, Porfirio; Koch, Stefan; Laukoetter, Mike G; Lee, Winston Y; Kolegraff, Keli; Capaldo, Christopher T; Beeman, Neal; Addis, Caroline; Gerner-Smidt, Kirsten; Neumaier, Irmgard; Skerra, Arne; Li, Linheng; Parkos, Charles A; Nusrat, Asma

    2010-01-01

    SUMMARY Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-γ (IFN-γ) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-β-catenin and Wingless-Int (Wnt)-β-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-γ resulted in activation of β-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-γ treatment, and reduced proliferation through depletion of the Wnt co-receptor LRP6. These effects were enhanced by tumor necrosis factor-α (TNF)-α, suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased β-catenin-T-cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-γ - deficient mice. Thus, we propose that IFN-γ regulates intestinal epithelial homeostasis by sequential regulation of converging β-catenin signaling pathways. PMID:20303298

  13. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4+ intestinal intraepithelial lymphocytes

    International Nuclear Information System (INIS)

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4+ IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4+ IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4+ 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+ 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+ LPLs and primed splenic CD4+ T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4+ IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo

  14. 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.

  15. Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells.

    Science.gov (United States)

    Ringqvist, Emma; Palm, J E Daniel; Skarin, Hanna; Hehl, Adrian B; Weiland, Malin; Davids, Barbara J; Reiner, David S; Griffiths, William J; Eckmann, Lars; Gillin, Frances D; Svärd, Staffan G

    2008-06-01

    Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine. PMID:18359106

  16. Permeability and modulation of the intestinal epithelial barrier in vitro

    OpenAIRE

    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 epithelium, however, renders the entrance of non-nutrient (potentially harmful) hydrophilic (macro-) molecules, viruses and bacteria into the systemic circulation very low by presenting an almost impermeable barrier ...

  17. Expansion of intestinal epithelial stem cells during murine development.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Dehmer

    Full Text Available Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs. Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.

  18. 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

  19. Rifaximin Improves Clostridium difficile Toxin A-Induced Toxicity in Caco-2 Cells by the PXR-Dependent TLR4/MyD88/NF-κB Pathway

    Science.gov (United States)

    Esposito, Giuseppe; Nobile, Nicola; Gigli, Stefano; Seguella, Luisa; Pesce, Marcella; d’Alessandro, Alessandra; Bruzzese, Eugenia; Capoccia, Elena; Steardo, Luca; Cuomo, Rosario; Sarnelli, Giovanni

    2016-01-01

    Background: Clostridium difficile infections (CDIs) caused by Clostridium difficile toxin A (TcdA) lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat. Aim: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action. Methods: Caco-2 cells were incubated with TcdA and treated with rifaximin (0.1-10 μM) with or without ketoconazole (10 μM). The transepithelial electrical resistance (TEER) and viability of the treated cells was determined. Also, the expression of zona occludens-1 (ZO-1), toll-like receptor 4 (TLR4), Bcl-2-associated X protein (Bax), transforming growth factor-β-activated kinase-1 (TAK1), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappaB (NF-κB) was determined. Results: Rifaximin treatment (0.1, 1.0, and 10 μM) caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360, 480, and 680% vs. TcdA treatment) 24 h after the treatment and improved their viability (61, 79, and 105%). Treatment also concentration-dependently decreased the expression of Bax protein (-29, -65, and -77%) and increased the expression of ZO-1 (25, 54, and 87%) and occludin (71, 114, and 262%) versus TcdA treatment. The expression of TLR4 (-33, -50, and -75%), MyD88 (-29, -60, and -81%) and TAK1 (-37, -63, and -79%) were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects. Conclusion: Rifaximin improved TcdA-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88/NF-κB pathway mechanism, and may be useful in the treatment of CDIs. PMID:27242527

  20. Lineage-specific expression of bestrophin-2 and bestrophin-4 in human intestinal epithelial cells

    DEFF Research Database (Denmark)

    Ito, Go; Okamoto, Ryuichi; Murano, Tatsuro;

    2013-01-01

    Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3(-) or Cl(-). Bestrophin genes represent a newly identified group of calcium-activated Cl(-) channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2...... (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes...

  1. Distinct Roles for Intestinal Epithelial Cell-Specific Hdac1 and Hdac2 in the Regulation of Murine Intestinal Homeostasis.

    Science.gov (United States)

    Gonneaud, Alexis; Turgeon, Naomie; Boudreau, François; Perreault, Nathalie; Rivard, Nathalie; Asselin, Claude

    2016-02-01

    The intestinal epithelium responds to and transmits signals from the microbiota and the mucosal immune system to insure intestinal homeostasis. These interactions are in part conveyed by epigenetic modifications, which respond to environmental changes. Protein acetylation is an epigenetic signal regulated by histone deacetylases, including Hdac1 and Hdac2. We have previously shown that villin-Cre-inducible intestinal epithelial cell (IEC)-specific Hdac1 and Hdac2 deletions disturb intestinal homeostasis. To determine the role of Hdac1 and Hdac2 in the regulation of IEC function and the establishment of the dual knockout phenotype, we have generated villin-Cre murine models expressing one Hdac1 allele without Hdac2, or one Hdac2 allele without Hdac1. We have also investigated the effect of short-term deletion of both genes in naphtoflavone-inducible Ah-Cre and tamoxifen-inducible villin-Cre(ER) mice. Mice with one Hdac1 allele displayed normal tissue architecture, but increased sensitivity to DSS-induced colitis. In contrast, mice with one Hdac2 allele displayed intestinal architecture defects, increased proliferation, decreased goblet cell numbers as opposed to Paneth cells, increased immune cell infiltration associated with fibrosis, and increased sensitivity to DSS-induced colitis. In comparison to dual knockout mice, intermediary activation of Notch, mTOR, and Stat3 signaling pathways was observed. While villin-Cre(ER) Hdac1 and Hdac2 deletions led to an impaired epithelium and differentiation defects, Ah-Cre-mediated deletion resulted in blunted proliferation associated with the induction of a DNA damage response. Our results suggest that IEC determination and intestinal homeostasis are highly dependent on Hdac1 and Hdac2 activity levels, and that changes in the IEC acetylome may alter the mucosal environment. PMID:26174178

  2. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration

    NARCIS (Netherlands)

    M. Verburg (Melissa); I.B. Renes (Ingrid); D.J. van Nispen; S. Ferdinandusse; M. Jorritsma; H.A. Büller (Hans); A.W.C. Einerhand (Sandra); J. Dekker (Jan)

    2002-01-01

    textabstractThe rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were loca

  3. Aggregation Substance Increases Adherence and Internalization, but Not Translocation, of Enterococcus faecalis through Different Intestinal Epithelial Cells In Vitro

    OpenAIRE

    Sartingen, S.; Rozdzinski, E; Muscholl-Silberhorn, A.; Marre, R

    2000-01-01

    The aggregation substance of Enterococcus faecalis increased bacterial adherence to and internalization by epithelial cells originating from the colon and duodenum but not by cells derived from the ileum. However, enterococcal translocation through monolayers of intestinal epithelium was not observed.

  4. 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....

  5. Gastric digestion of pea ferritin and modulation of its iron bioavailability by ascorbic and phytic acids in caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Satyanarayana Bejjani; Raghu Pullakhandam; Ravinder Punjal; K Madhavan Nair

    2007-01-01

    AIM: To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model.METHODS: Pea seed ferritin was purified using salt fractionation followed by gel filtration chromatography.The bioavailability of ferritin iron was assessed using coupled in vitro digestion/Caco-2 cell model in the presence or absence of ascorbic acid and phytic acid.Caco-2 cell ferritin formation was used as a surrogate marker of iron uptake. Structural changes of pea ferritin under simulated gastric pH were characterized using electrophoresis, gel filtration and circular dichroism spectroscopy.RESULTS: The caco-2 cell ferritin formation was significantly increased (P < 0.001) with FeSO4 (19.3±9.8 ng/mg protein) and pea ferritin (13.9 ± 6.19 ng/mg protein) compared to the blank digest (3.7 ± 1.8 ng/mg protein). Ascorbic acid enhanced while phytic acid decreased the pea ferritin iron bioavailability. However,either in the presence or absence of ascorbic acid, the ferritin content of caco-2 cells was significantly less with pea ferritin than with FeSO4. At gastric pH, no band corresponding to ferritin was observed in the presence of pepsin either on native PAGE or SDS-PAGE. Gel filtration chromatography and circular dichroism spectroscopy revealed a pH dependent loss of quaternary and secondary structure.CONCLUSION: Under gastric conditions, the iron core of pea ferritin is released into the digestive medium due to acid induced structural alterations and dissociation of protein. The released iron interacts with dietary factors leading to modulation of pea ferritin iron bioavailability,resembling the typical characteristics of non-heme iron.

  6. 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.

  7. 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...

  8. Enteric glia cells attenuate cytomix-induced intestinal epithelial barrier breakdown.

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    Gerald A Cheadle

    Full Text Available BACKGROUND: Intestinal barrier failure may lead to systemic inflammation and distant organ injury in patients following severe injury. Enteric glia cells (EGCs have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO. We have recently shown than Vagal Nerve Stimulation (VNS increases EGC activation, which was associated with improved gut barrier integrity. Thus, we sought to further study the mechanism by which EGCs prevent intestinal barrier breakdown utilizing an in vitro model. We postulated that EGCs, through the secretion of GSNO, would improve intestinal barrier function through improved expression and localization of intestinal tight junction proteins. METHODS: Epithelial cells were co-cultured with EGCs or incubated with GSNO and exposed to Cytomix (TNF-α, INF-γ, IL-1β for 24 hours. Barrier function was assessed by permeability to 4kDa FITC-Dextran. Changes in tight junction proteins ZO-1, occludin, and phospho-MLC (P-MLC were assessed by immunohistochemistry and immunoblot. KEY RESULTS: Co-culture of Cytomix-stimulated epithelial monolayers with EGCs prevented increases in permeability and improved expression and localization of occludin, ZO-1, and P-MLC. Further, treatment of epithelial monolayers with GSNO also prevented Cytomix-induced increases in permeability and exhibited a similar improvement in expression and localization of occludin, ZO-1, and P-MLC. CONCLUSIONS & INFERENCES: The addition of EGCs, or their secreted mediator GSNO, prevents epithelial barrier failure after injury and improved expression of tight junction proteins. Thus, therapies that increase EGC activation, such as VNS, may be a novel strategy to limit barrier failure in patients following severe injury.

  9. Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine

    OpenAIRE

    Vyas, D.; Robertson, C M; Stromberg, P.E.; J. R. Martin; Dunne, W. M.; Houchen, C.W.; Barrett, T A; Ayala, A; Perl, M.; Buchman, T G; Coopersmith, C.M.

    2007-01-01

    Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal...

  10. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

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    Amol Bhargava

    Full Text Available Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1, suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK. Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  11. ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation

    Science.gov (United States)

    de Jong, Petrus R.; Taniguchi, Koji; Harris, Alexandra R.; Bertin, Samuel; Takahashi, Naoki; Duong, Jen; Campos, Alejandro D.; Powis, Garth; Corr, Maripat; Karin, Michael; Raz, Eyal

    2016-01-01

    The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC. PMID:27187615

  12. Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

    Science.gov (United States)

    Koch, Stefan; Capaldo, Christopher T.; Samarin, Stanislav; Nava, Porfirio; Neumaier, Irmgard; Skerra, Arne; Sacks, David B.; Parkos, Charles A.

    2009-01-01

    Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo. PMID:19776352

  13. Food contaminant zearalenone and its metabolites affect cytokine synthesis and intestinal epithelial integrity of porcine cells.

    Science.gov (United States)

    Marin, Daniela E; Motiu, Monica; Taranu, Ionelia

    2015-06-01

    The intestinal epithelium is the first barrier against food contaminants. Zearalenone (ZEN) is an estrogenic mycotoxin that was identified as a common contaminant of cereal grains and food and feedstuffs. In the present study, we have investigated the in vitro effects of ZEN and some of its metabolites (α-ZOL, β-ZOL) in concentrations of 10-100 µM on a swine epithelial cell line: Intestinal porcine epithelial cells (IPEC-1). We demonstrated that both ZEN metabolites were more toxic for IPEC cells as resulted from the XTT test, while for doses lower than 10 µM, only β-ZOL showed a more pronounced cytotoxicity versus epithelial cells as resulted from neutral red assay. ZEN has no effect on TER values, while α-ZOL significantly decreased the TER values, starting with day 4 of treatment. β-ZOL had a dual effect, firstly it induced a significant increase of TER, and then, starting on day 6, it induced a dramatic decrease of TER values as compared with on day 0. Concerning the cytokine synthesis, our results showed that ZEN has a tendency to increase the synthesis of IL-8 and IL-10. By contrast, α- and β-ZOL decreased the expression of both IL-8 and IL-10, in a dose dependent manner. In conclusion, our results showed that ZEN and its metabolites differently affected porcine intestinal cell viability, transepithelial resistance and cytokine synthesis with important implication for gut health. PMID:26035492

  14. Suitability of the in vitro Caco-2 assay to predict the oral absorption of aromatic amine hair dyes.

    Science.gov (United States)

    Obringer, Cindy; Manwaring, John; Goebel, Carsten; Hewitt, Nicola J; Rothe, Helga

    2016-04-01

    Oral absorption is a key element for safety assessments of cosmetic ingredients, including hair dye molecules. Reliable in vitro methods are needed since the European Union has banned the use of animals for the testing of cosmetic ingredients. Caco-2 cells were used to measure the intestinal permeability characteristics (Papp) of 14 aromatic amine hair dye molecules with varying chemical structures, and the data were compared with historical in vivo oral absorption rat data. The majority of the hair dyes exhibited Papp values that indicated good in vivo absorption. The moderate to high oral absorption findings, i.e. ≥60%, were confirmed in in vivo rat studies. Moreover, the compound with a very low Papp value (APB: 3-((9,10-dihydro-9,10-dioxo-4-(methylamino)-1-anthracenyl)amino)-N,N-dimethyl-N-propyl-1-propanaminium) was poorly absorbed in vivo as well (5% of the dose). This data set suggests that the Caco-2 cell model is a reliable in vitro tool for the determination of the intestinal absorption of aromatic amines with diverse chemical structures. When used in combination with other in vitro assays for metabolism and skin penetration, the Caco-2 model can contribute to the prediction and mechanistic interpretation of the absorption, metabolism and elimination properties of cosmetic ingredients without the use of animals. PMID:26578466

  15. Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

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    Dismuke Adria D

    2009-07-01

    Full Text Available Abstract Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo.

  16. Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

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    Kohn Aimee

    2009-01-01

    Full Text Available Abstract Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo.

  17. Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells.

    Science.gov (United States)

    Rimoldi, Monica; Chieppa, Marcello; Salucci, Valentina; Avogadri, Francesca; Sonzogni, Angelica; Sampietro, Gianluca M; Nespoli, Angelo; Viale, Giuseppe; Allavena, Paola; Rescigno, Maria

    2005-05-01

    The control of damaging inflammation by the mucosal immune system in response to commensal and harmful ingested bacteria is unknown. Here we show epithelial cells conditioned mucosal dendritic cells through the constitutive release of thymic stromal lymphopoietin and other mediators, resulting in the induction of 'noninflammatory' dendritic cells. Epithelial cell-conditioned dendritic cells released interleukins 10 and 6 but not interleukin 12, and they promoted the polarization of T cells toward a 'classical' noninflammatory T helper type 2 response, even after exposure to a T helper type 1-inducing pathogen. This control of immune responses seemed to be lost in patients with Crohn disease. Thus, the intimate interplay between intestinal epithelial cells and dendritic cells may help to maintain gut immune homeostasis. PMID:15821737

  18. Intestinal epithelial function and villus surface area in rats with bile fistulae

    International Nuclear Information System (INIS)

    We have previously found reduced absorption of vitamin B-12 in rats with choledochocolic fistulae. To investigate whether the reduction is caused by epithelial dysfunction or mucosal hypoplasia, choledocholic fistulae were made in 11 rats, whereas 10 rats were sham-operated. The epithelial function was evaluated 9 days later by measuring the uptake of 57CoB 1 2 and glucose in perfused intestinal segments, and by determining the activities of 11 mucosal enzymes. Hypoplasia was investigated by performing morphometric measurements of the villus surface area and by measuring the weight, protein, and DNA in mucosal scrapings. The results suggest that choledochocolic fistulae in rats do not impair epithelial function or cause mucosal hypoplasia. The urinary excretion of indican was increased in the fistula-operated rats, but further studies are needed to establish the significance of this observation

  19. Lactobacillus plantarum CUL66 can impact cholesterol homeostasis in Caco-2 enterocytes.

    Science.gov (United States)

    Michael, D R; Moss, J W E; Calvente, D Lama; Garaiova, I; Plummer, S F; Ramji, D P

    2016-06-01

    Hypercholesterolemia drives the development of cardiovascular disease, the leading cause of mortality in western society. Supplementation with probiotics that interfere with cholesterol metabolism may provide a contribution to disease prevention. Lactobacillus plantarum CUL66 (NCIMB 30280) has been assessed in vitro for its ability to impact cholesterol absorption. L. plantarum CUL66 tested positive for bile salt hydrolase activity and the ability to assimilate cholesterol from culture media. RT-qPCR analysis showed that the bacterium significantly decreased the expression of Niemann-Pick C1-like 1 and ATP-binding cassette transporter-1 in polarised Caco-2 cells after 6 h exposure. Conversely, the expression of ATP-binding cassette sub-family G member (ABCG)-5 and ABCG-8, and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased. Using a radiolabelled assay, we also observed significant reductions in the uptake and basolateral efflux of cholesterol by Caco-2 cells exposed to L. plantarum CUL66. This in vitro study identified L. plantarum CUL66 as a cholesterol lowering bacteria by highlighting its ability to beneficially regulate multiple in vitro events associated with intestinal cholesterol metabolism and provides evidence of efficacy for its inclusion in future in vivo studies. PMID:26839071

  20. Heme in intestinal epithelial cell turnover, differentiation,detoxification, inflammation, carcinogenesis, absorption and motility

    Institute of Scientific and Technical Information of China (English)

    Phillip S Oates; Adrian R West

    2006-01-01

    The gastrointestinal tract is lined by a simple epithelium that undergoes constant renewal involving cell division,differentiation and cell death. In addition, the epithelial lining separates the hostile processes of digestion and absorption that occur in the intestinal lumen from the aseptic environment of the internal milieu by defensive mechanisms that protect the epithelium from being breached. Central to these defensive processes is the synthesis of heme and its catabolism by heme oxygenase (HO). Dietary heme is also an important source of iron for the body which is taken up intact by the enterocyte.This review describes the recent literature on the diverse properties of heme/HO in the intestine tract.The roles of heme/HO in the regulation of the cell cycle/apoptosis, detoxification of xenobiotics, oxidative stress,inflammation, development of colon cancer, hemeiron absorption and intestinal motility are specifically examined.

  1. Hydrogen peroxide generation in caco-2 cell culture medium by addition of phenolic compounds: effect of ascorbic acid.

    Science.gov (United States)

    Roques, Sylvie Cambon; Landrault, Nicolas; Teissèdre, Pierre-Louis; Laurent, Caroline; Besançon, Pierre; Rouane, Jean-Max; Caporiccio, Bertrand

    2002-05-01

    Phenolic compounds have recently attracted special attention due to their beneficial health effects; their intestinal absorption and bioavailability need, therefore, to be investigated and Caco-2 cell culture model appeared as a promising tool. We have shown herein that the addition of a grape seed extract (GSE) to Dulbecco's modified Eagle's medium (DMEM) used for Caco-2 cell culture leads to a substantial loss of catechin, epicatechin and B2 and B3 dimers from GSE in the medium after 24 h and to a production of hydrogen peroxide (H2O2). When 1420 microM ascorbic acid is added to the DMEM, such H2O2 production was prevented. This hydrogen peroxide generation substantially involves inorganic salts from the DMEM. We recommend that ascorbic acid be added to circumvent such a risk. PMID:12150547

  2. Cell dedifferentiation and epithelial to mesenchymal transitions during intestinal regeneration in H. glaberrima

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    Rivera-Cruz Angélica

    2011-10-01

    Full Text Available Abstract Background Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima. Results We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes. Conclusions Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.

  3. Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells

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    Basora, Nuria, E-mail: Nuria.Basora@USherbrooke.ca [Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1N 5N4 (Canada); Tetreault, Marie-Pier; Boucher, Marie-Pierre; Herring, Elizabeth; Beaulieu, Jean-Francois [Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1N 5N4 (Canada)

    2010-05-15

    In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the {beta}-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.

  4. Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells

    International Nuclear Information System (INIS)

    In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the β-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.

  5. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    International Nuclear Information System (INIS)

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2-3H]mannose or L-[5,6-3H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2-3H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2-3H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6-3H]glucosamine and L-[1-14C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3H-labeled N-acetylglucosamine and N-acetylgalactosamine

  6. Functional expression of double-stranded RNA-dependent protein kinase in rat intestinal epithelial cells.

    Science.gov (United States)

    Sato, Nagahiro; Morimoto, Hiroyuki; Baba, Ryoko; Nakamata, Junichi; Doi, Yoshiaki; Yamaguchi, Koji

    2010-05-01

    Intestinal epithelial cells (IECs) are exposed to external environment, microbial and viral products, and serve as essential barriers to antigens. Recent studies have shown that IECs express Toll-like receptors (TLRs) and respond to microbial components. The antimicrobial and antiviral barriers consist of many molecules including TLRs. To investigate the further component of this barrier in intestine, we examined the expression of double-stranded RNA-dependent protein kinase (PKR). PKR is a player in the cellular antiviral response and phosphorylates alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha) to block protein synthesis and induces apoptosis. In this study, we showed that the expression of PKR was restricted to the cytoplasm of absorptive epithelial cells in the intestine of adult rat. We also demonstrated that PKR was expressed in the cultured rat intestinal epithelial cells (IEC-6). The level of PKR protein expression and the activity of alkaline phosphatase (ALP) increased in the cultured IEC-6 cells in a time-dependent manner. Inhibition of PKR by the 2-aminopurine treatment decreased ALP activity in the IEC-6 cells. Treatment of IEC-6 cells with synthetic double-stranded RNA (dsRNA) induced cell death in a dose-dependent manner. The addition of hydrocortisone also provoked suppression of PKR expression and ALP activity. This modulation might be mediated by signal transducers and activators of transcription-1 (STAT-1) protein. We concluded that PKR is expressed in IECs as potent barriers to antigens and is a possible modulator of the differentiation of rat IECs. PMID:20213745

  7. Histological alterations of intestinal villi and epithelial cells after feeding dietary sugar cane extract in piglets

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    Toshikazu Kawai

    2012-07-01

    Full Text Available Effects of sugar cane extract (SCE on the piglet intestinal histology were observed. Twelve castrated male piglets weaned at the age of 26 days were allotted to three groups fed diets containing 0, 0.05 or 0.10% SCE. At the end of feeding experiment, each intestinal segment was taken for light or scanning electron microscopy. Feed intake, body weight gain and feed efficiency did not show a difference among groups. Most of the values for villus height, villus area, cell area and cell mitosis numbers were not different among groups, except for that the villus area of the 0.10% SCE group and the cell area of both SCE groups increased significantly at the jejunum compared to the control (P<0.05. For cell mitosis numbers, the 0.10% SCE group was higher than the 0.05% SCE group at the jejunum. Compared with the majority of flat cells of each intestinal segment in the control, the SCE groups had protuberated cells. In the 0.05% SCE group, deeper cells at the sites of recently exfoliated cells in the duodenum, cell clusters aggregated by protuberated cells in the jejunum and much more protuberant cells in the ileum were observed. These histological intestinal alterations suggest that SCE could raise the functions of intestinal villi and epithelial cells, especially at the 0.05%.

  8. Promotion of Intestinal Epithelial Cell Turnover by Commensal Bacteria: Role of Short-Chain Fatty Acids.

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    Jung-Ha Park

    Full Text Available The life span of intestinal epithelial cells (IECs is short (3-5 days, and its regulation is thought to be important for homeostasis of the intestinal epithelium. We have now investigated the role of commensal bacteria in regulation of IEC turnover in the small intestine. The proliferative activity of IECs in intestinal crypts as well as the migration of these cells along the crypt-villus axis were markedly attenuated both in germ-free mice and in specific pathogen-free (SPF mice treated with a mixture of antibiotics, with antibiotics selective for Gram-positive bacteria being most effective in this regard. Oral administration of chloroform-treated feces of SPF mice to germ-free mice resulted in a marked increase in IEC turnover, suggesting that spore-forming Gram-positive bacteria contribute to this effect. Oral administration of short-chain fatty acids (SCFAs as bacterial fermentation products also restored the turnover of IECs in antibiotic-treated SPF mice as well as promoted the development of intestinal organoids in vitro. Antibiotic treatment reduced the phosphorylation levels of ERK, ribosomal protein S6, and STAT3 in IECs of SPF mice. Our results thus suggest that Gram-positive commensal bacteria are a major determinant of IEC turnover, and that their stimulatory effect is mediated by SCFAs.

  9. Promotion of Intestinal Epithelial Cell Turnover by Commensal Bacteria: Role of Short-Chain Fatty Acids.

    Science.gov (United States)

    Park, Jung-Ha; Kotani, Takenori; Konno, Tasuku; Setiawan, Jajar; Kitamura, Yasuaki; Imada, Shinya; Usui, Yutaro; Hatano, Naoya; Shinohara, Masakazu; Saito, Yasuyuki; Murata, Yoji; Matozaki, Takashi

    2016-01-01

    The life span of intestinal epithelial cells (IECs) is short (3-5 days), and its regulation is thought to be important for homeostasis of the intestinal epithelium. We have now investigated the role of commensal bacteria in regulation of IEC turnover in the small intestine. The proliferative activity of IECs in intestinal crypts as well as the migration of these cells along the crypt-villus axis were markedly attenuated both in germ-free mice and in specific pathogen-free (SPF) mice treated with a mixture of antibiotics, with antibiotics selective for Gram-positive bacteria being most effective in this regard. Oral administration of chloroform-treated feces of SPF mice to germ-free mice resulted in a marked increase in IEC turnover, suggesting that spore-forming Gram-positive bacteria contribute to this effect. Oral administration of short-chain fatty acids (SCFAs) as bacterial fermentation products also restored the turnover of IECs in antibiotic-treated SPF mice as well as promoted the development of intestinal organoids in vitro. Antibiotic treatment reduced the phosphorylation levels of ERK, ribosomal protein S6, and STAT3 in IECs of SPF mice. Our results thus suggest that Gram-positive commensal bacteria are a major determinant of IEC turnover, and that their stimulatory effect is mediated by SCFAs. PMID:27232601

  10. Expression of integrin alphavbeta6 in the intestinal epithelial cells of patients with inflammatory bowel disease

    Directory of Open Access Journals (Sweden)

    Bai-Sui Feng

    2009-09-01

    Full Text Available Background and aims: The prevalence of inflammatory bowel disease (IBD is about 0.05% in industrialized countries. The pathogenesis of IBD remains to be further understood. The present study aims to elucidate the expression of integrin αvβ6 in the intestinal mucosa of patients with IBD. Materials and Methods: Colonic biopsy was obtained from a group of IBD patients. The expression of αvβ6 in the intestinal mucosa was detected by Western blotting. Human colonic epithelial cell line T84 cells were stimulated by microbial antigen flagellin. The expression of αvβ6 in T84 cells was evaluated by quantitative RT-PCR and Western blotting. Results: The levels of αvβ6 in the intestinal mucosa were much lower than it in normal control subjects. The serum levels of myeloperoxidase (MPO were higher in IBD patients that were negatively correlated with the levels of αvβ6 in the intestinal mucosa. The expression of αvβ6 was detectable in T84 cells at naïve status that could be upregulated by exposure to microbial antigen flagellin. Pretreatment with MPO dramatically suppressed the expression of αvβ6 in T84 cells. Conclusions: We conclude that the expression of αvβ6 was suppressed in IBD intestinal mucosa, which could be resulted from the high levels of MPO.

  11. Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG

    OpenAIRE

    Burkholder Kristin M; Bhunia Arun K

    2009-01-01

    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...

  12. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

    Science.gov (United States)

    Qin, Ying; Naito, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2011-11-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic

  13. Arginine Consumption by the Intestinal Parasite Giardia intestinalis Reduces Proliferation of Intestinal Epithelial Cells

    OpenAIRE

    Stadelmann, Britta; Merino, Maria C.; Persson, Lo; Svard, Staffan G.

    2012-01-01

    In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the host´s 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 consu...

  14. Protective effects of ID331 Triticum monococcum gliadin on in vitro models of the intestinal epithelium.

    Science.gov (United States)

    Iacomino, Giuseppe; Di Stasio, Luigia; Fierro, Olga; Picariello, Gianluca; Venezia, Antonella; Gazza, Laura; Ferranti, Pasquale; Mamone, Gianfranco

    2016-12-01

    A growing interest in developing new strategies for preventing coeliac disease has motivated efforts to identify cereals with null or reduced toxicity. In the current study, we investigate the biological effects of ID331 Triticum monococcum gliadin-derived peptides in human Caco-2 intestinal epithelial cells. Triticum aestivum gliadin derived peptides were employed as a positive control. The effects on epithelial permeability, zonulin release, viability, and cytoskeleton reorganization were investigated. Our findings confirmed that ID331 gliadin did not enhance permeability and did not induce zonulin release, cytotoxicity or cytoskeleton reorganization of Caco-2 cell monolayers. We also demonstrated that ID331 ω-gliadin and its derived peptide ω(105-123) exerted a protective action, mitigating the injury of Triticum aestivum gliadin on cell viability and cytoskeleton reorganization. These results may represent a new opportunity for the future development of innovative strategies to reduce gluten toxicity in the diet of patients with gluten intolerance. PMID:27374565

  15. Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis.

    Science.gov (United States)

    Perrone, Erin E; Jung, Enjae; Breed, Elise; Dominguez, Jessica A; Liang, Zhe; Clark, Andrew T; Dunne, W Michael; Burd, Eileen M; Coopersmith, Craig M

    2012-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus-induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid-/- mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand-/- mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant. PMID:22592747

  16. Functionalized carbon nanomaterials: exploring the interactions with Caco-2 cells for potential oral drug delivery

    Directory of Open Access Journals (Sweden)

    Coyuco JC

    2011-10-01

    Full Text Available Jurja C Coyuco, Yuanjie Liu, Bee-Jen Tan, Gigi NC ChiuDepartment of Pharmacy, Faculty of Science, National University of Singapore, SingaporeAbstract: 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 (fSWCNT-COOH and poly(ethylene glycol functionalized single-walled carbon nanotubes (fSWCNT-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, fSWCNT-COOH significantly reduced transepithelial electrical resistance and enhanced transport of Lucifer Yellow across the Caco-2 monolayer. Confocal fluorescence microscopy showed that fSWCNT-COOH treated cells had the highest perturbation in the distribution of ZO-1, a protein marker of tight junction, suggesting that fSWCNT-COOH could enhance paracellular permeability via disruption of tight junctions. This modulating effect of fSWCNT-COOH can be reversed over time. Furthermore, cellular accumulation of the P-gp substrate, rhodamine-123, was significantly increased in cells treated with fSWCNT-COOH, suggestive of P-gp inhibition. Of note, fSWCNT-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

  17. Cross-talk between intestinal epithelial cells and immune cells in inflammatory bowel disease

    Science.gov (United States)

    Al-Ghadban, Sara; Kaissi, Samira; Homaidan, Fadia R.; Naim, Hassan Y.; El-Sabban, Marwan E.

    2016-01-01

    Inflammatory bowel disease (IBD) involves functional impairment of intestinal epithelial cells (IECs), concomitant with the infiltration of the lamina propria by inflammatory cells. We explored the reciprocal paracrine and direct interaction between human IECs and macrophages (MΦ) in a co-culture system that mimics some aspects of IBD. We investigated the expression of intercellular junctional proteins in cultured IECs under inflammatory conditions and in tissues from IBD patients. IECs establish functional gap junctions with IECs and MΦ, respectively. Connexin (Cx26) and Cx43 expression in cultured IECs is augmented under inflammatory conditions; while, Cx43-associated junctional complexes partners, E-cadherin, ZO-1, and β-catenin expression is decreased. The expression of Cx26 and Cx43 in IBD tissues is redistributed to the basal membrane of IEC, which is associated with decrease in junctional complex proteins’ expression, collagen type IV expression and infiltration of MΦ. These data support the notion that the combination of paracrine and hetero-cellular communication between IECs and MΦs may regulate epithelial cell function through the establishment of junctional complexes between inflammatory cells and IECs, which ultimately contribute to the dys-regulation of intestinal epithelial barrier. PMID:27417573

  18. The impact of hypoxia on intestinal epithelial cell functions: consequences for invasion by bacterial pathogens.

    Science.gov (United States)

    Zeitouni, Nathalie E; Chotikatum, Sucheera; von Köckritz-Blickwede, Maren; Naim, Hassan Y

    2016-12-01

    The maintenance of oxygen homeostasis in human tissues is mediated by several cellular adaptations in response to low-oxygen stress, called hypoxia. A decrease in tissue oxygen levels is initially counteracted by increasing local blood flow to overcome diminished oxygenation and avoid hypoxic stress. However, studies have shown that the physiological oxygen concentrations in several tissues are much lower than atmospheric (normoxic) conditions, and the oxygen supply is finely regulated in individual cell types. The gastrointestinal tract has been described to subsist in a state of physiologically low oxygen level and is thus depicted as a tissue in the state of constant low-grade inflammation. The intestinal epithelial cell layer plays a vital role in the immune response to inflammation and infections that occur within the intestinal tissue and is involved in many of the adaptation responses to hypoxic stress. This is especially relevant in the context of inflammatory disorders, such as inflammatory bowel disease (IBD). Therefore, this review aims to describe the intestinal epithelial cellular response to hypoxia and the consequences for host interactions with invading gastrointestinal bacterial pathogens. PMID:27002817

  19. Epithelial IL-23R Signaling Licenses Protective IL-22 Responses in Intestinal Inflammation.

    Science.gov (United States)

    Aden, Konrad; Rehman, Ateequr; Falk-Paulsen, Maren; Secher, Thomas; Kuiper, Jan; Tran, Florian; Pfeuffer, Steffen; Sheibani-Tezerji, Raheleh; Breuer, Alexandra; Luzius, Anne; Jentzsch, Marlene; Häsler, Robert; Billmann-Born, Susanne; Will, Olga; Lipinski, Simone; Bharti, Richa; Adolph, Timon; Iovanna, Juan L; Kempster, Sarah L; Blumberg, Richard S; Schreiber, Stefan; Becher, Burkhard; Chamaillard, Mathias; Kaser, Arthur; Rosenstiel, Philip

    2016-08-23

    A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells. PMID:27524624

  20. High therapeutic efficacy of Cathelicidin-WA against postweaning diarrhea via inhibiting inflammation and enhancing epithelial barrier in the intestine

    Science.gov (United States)

    Yi, Hongbo; Zhang, Lin; Gan, Zhenshun; Xiong, Haitao; Yu, Caihua; Du, Huahua; Wang, Yizhen

    2016-01-01

    Diarrhea is a leading cause of death among young mammals, especially during weaning. Here, we investigated the effects of Cathelicidin-WA (CWA) on diarrhea, intestinal morphology, inflammatory responses, epithelial barrier and microbiota in the intestine of young mammals during weaning. Piglets with clinical diarrhea were selected and treated with saline (control), CWA or enrofloxacin (Enro) for 4 days. Both CWA and Enro effectively attenuated diarrhea. Compared with the control, CWA decreased IL-6, IL-8 and IL-22 levels and reduced neutrophil infiltration into the jejunum. CWA inhibited inflammation by down-regulating the TLR4-, MyD88- and NF-κB-dependent pathways. Additionally, CWA improved intestinal morphology by increasing villus and microvillus heights and enhancing intestinal barrier function by increasing tight junction (TJ) protein expression and augmenting wound-healing ability in intestinal epithelial cells. CWA also improved microbiota composition and increased short-chain fatty acid (SCFA) levels in feces. By contrast, Enro not only disrupted the intestinal barrier but also negatively affected microbiota composition and SCFA levels in the intestine. In conclusion, CWA effectively attenuated inflammation, enhanced intestinal barrier function, and improved microbiota composition in the intestines of weaned piglets. These results suggest that CWA could be an effective and safe therapy for diarrhea or other intestinal diseases in young mammals. PMID:27181680

  1. Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study.

    Science.gov (United States)

    Bianco, Giuseppe; Fontanella, Bianca; Severino, Lorella; Quaroni, Andrea; Autore, Giuseppina; Marzocco, Stefania

    2012-01-01

    The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5-80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G(0)/G(1) interphase and in the S phase, with a concomitant reduction in the fraction of cells in G(2). Interestingly, when administered at lower concentrations (0.1-2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins. PMID:23251682

  2. Carrageenan Induces Cell Cycle Arrest in Human Intestinal Epithelial Cells in Vitro1–3

    Science.gov (United States)

    Bhattacharyya, Sumit; Borthakur, Alip; Dudeja, Pradeep K.; Tobacman, Joanne K.

    2016-01-01

    Multiple studies in animal models have shown that the commonly used food additive carrageenan (CGN) induces inflammation and intestinal neoplasia. We performed the first studies to determine the effects of CGN exposure on human intestinal epithelial cells (IEC) in tissue culture and tested the effect of very low concentrations (1–10 mg/L) of undegraded, high-molecular weight CGN. These concentrations of CGN are less than the anticipated exposure of the human colon to CGN from the average Western diet. In the human colonic epithelial cell line NCM460 and in primary human colonic epithelial cells that were exposed to CGN for 1–8 d, we found increased cell death, reduced cell proliferation, and cell cycle arrest compared with unexposed control cells. After 6–8 d of CGN exposure, the percentage of cells reentering G0–G1 significantly decreased and the percentages of cells in S and G2-M phases significantly increased. Increases in activated p53, p21, and p15 followed CGN exposure, consistent with CGN-induced cell cycle arrest. Additional data, including DNA ladder, poly ADP ribose polymerase Western blot, nuclear DNA staining, and activities of caspases 3 and 7, indicated no evidence of increased apoptosis following CGN exposure and were consistent with CGN-induced necrotic cell death. These data document for the first time, to our knowledge, marked adverse effects of low concentrations of CGN on survival of normal human IEC and suggest that CGN exposure may have a role in development of human intestinal pathology. PMID:18287351

  3. Heparin stimulates the proliferation of intestinal epithelial cells in primary culture.

    Science.gov (United States)

    Flint, N; Cove, F L; Evans, G S

    1994-02-01

    Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminoglycans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysaccharides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration. PMID:8207071

  4. Immunopathology of giardiasis: the role of lymphocytes in intestinal epithelial injury and malfunction

    Directory of Open Access Journals (Sweden)

    AG Buret

    2005-03-01

    Full Text Available T lymphocyte-mediated pathogenesis is common to a variety of enteropathies, including giardiasis, cryptosporidiosis, bacterial enteritis, celiac's disease, food anaphylaxis, and Crohn's disease. In giardiasis as well as in these other disorders, a diffuse loss of microvillous brush border, combined or not with villus atrophy, is responsible for disaccharidase insufficiencies and malabsorption of electrolytes, nutrients, and water, which ultimately cause diarrheal symptoms. Other mucosal changes may include crypt hyperplasia and increased infiltration of intra-epithelial lymphocytes. Recent studies using models of giardiasis have shed new light on the immune regulation of these abnormalities. Indeed, experiments using an athymic mouse model of infection have found that these epithelial injuries were T cell-dependent. Findings from further research indicate that that the loss of brush border surface area, reduced disaccharidase activities, and increase crypt-villus ratios are mediated by CD8+ T cells, whereas both CD8+ and CD4+ small mesenteric lymph node T cells regulate the influx of intra-epithelial lymphocytes. Future investigations need to characterize the CD8+ T cell signaling cascades that ultimately lead to epithelial injury and malfunction in giardiasis and other malabsorptive disorders of the intestine.

  5. Phytic acid protects porcine intestinal epithelial cells from deoxynivalenol (DON) cytotoxicity.

    OpenAIRE

    Pacheco, Graziela Drociunas; Silva, Caio Abércio da; Pinton, Philippe; Oswald, Isabelle

    2012-01-01

    The purpose of this study was to evaluate the effects of phytic acid (IP(6)) as a possible inhibitor of cellular damage induced by toxic substances such as mycotoxins on a porcine intestinal epithelial cell line (IPEC-1). We first observed that a dose of 5 mM phytic acid decreases cell viability and transepithelial electrical resistance (TEER) of cell monolayer. We next investigate the effect of non-cytotoxic dose of phytic acid on the deoxinivalenol (DON) induced decreased TEER. We showed th...

  6. Transforming growth factor-beta mediates intestinal healing and susceptibility to injury in vitro and in vivo through epithelial cells.

    Science.gov (United States)

    Beck, Paul L; Rosenberg, Ian M; Xavier, Ramnik J; Koh, Theodore; Wong, Josée F; Podolsky, Daniel K

    2003-02-01

    In vitro studies suggest that transforming growth factor (TGF)-beta has potent effects on gastrointestinal mucosal integrity, wound repair, and neoplasia. However, the multiplicity of actions of this peptide on many different cell types confounds efforts to define the role of TGF-beta within the intestinal epithelium in vivo. To delineate these effects selective blockade of intestinal epithelial TGF-beta activity was undertaken through targeted expression of a dominant-negative (DN) TGF-beta RII to intestinal epithelial cells in vitro and in vivo. Stable intestinal epithelial cell (IEC)-6 lines overexpressing TGF-beta RII-DN (nucleotides -7 to 573) were established. Transgenic mice overexpressing TGF-beta RII-DN under the regulation of a modified liver fatty acid-binding promoter (LFABP-PTS4) were constructed. In vitro healing was assessed by wounding of confluent monolayers. Colitis was induced by the addition of dextran sodium sulfate (2.5 to 7.5% w/v) to their drinking water. Overexpression of TGF-beta RII-DN in intestinal epithelial cell-6 cells resulted in a marked reduction in cell migration and TGF-beta-stimulated wound healing in vitro. TGF-beta RII-DN transgenic mice did not exhibit baseline intestinal inflammation or changes in survival, body weight, epithelial cell proliferation, aberrant crypt foci, or tumor formation. TGF-beta RII-DN mice were markedly more susceptible to dextran sodium sulfate-induced colitis and exhibited impaired recovery after colonic injury. TGF-beta is required for intestinal mucosal healing and TGF-beta modulation of the intestinal epithelium plays a central role in determining susceptibility to injury. PMID:12547717

  7. The Ets dominant repressor En/Erm enhances intestinal epithelial tumorigenesis in ApcMin mice

    International Nuclear Information System (INIS)

    Ets transcription factors have been widely implicated in the control of tumorigenesis, with most studies suggesting tumor-promoting roles. However, few studies have examined Ets tumorigenesis-modifying functions in vivo using model genetic systems. Using mice expressing a previously characterized Ets dominant repressor transgene in the intestinal epithelium (Villin-En/Erm), we examined the consequences of blocking endogenous Ets-mediated transcriptional activation on tumorigenesis in the ApcMin model of intestinal carcinoma. En/Erm expression in the intestine, at levels not associated with overt crypt-villus dysmorphogenesis, results in a marked increase in tumor number in ApcMin animals. Moreover, when examined histologically, tumors from En/Erm-expressing animals show a trend toward greater stromal invasiveness. Detailed analysis of crypt-villus homeostasis in these En/Erm transgenic animals suggests increased epithelial turnover as one possible mechanism for the enhanced tumorigenesis. Our findings provide in vivo evidence for a tumor-restricting function of endogenous Ets factors in the intestinal epithelium

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

    Directory of Open Access Journals (Sweden)

    Benoît Couvigny

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

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

    Science.gov (United States)

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

    2015-01-01

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

  10. Benzotropolone moiety in theaflavins is responsiblefor inhibitingpeptide-transport and activating AMP-activated protein kinase in Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Ha-Young Park

    2013-05-01

    Full Text Available ABSTRACTObjective:In the small intestine, peptide transporter 1 (PEPT1 plays a role in the transport of di- and tri-peptides. Recently, we found that theaflavins (TFs, dimeric catechins, inhibitedthe transport of di-peptides across Caco-2 monolayersby suppressingthe expression of PEPT1 through AMP-activated protein kinase (AMPK activation. In this study, we investigated the structural requirement of theaflavinsfor the effect, and the mechanism(sunderling theaflavin-induced AMPK activation.Methods:Theaflavin-3’-O-gallate (TF3’G was used forthis study, since it possessed the most potent inhibition power for peptide-transport among theaflavins. Absorption ability was measured with Caco-2 cell monolayers treated with or without 20 M sample (TF3’G or its related compounds in an Ussing Chamber. The amountof Gly-Sar (a model of PEPT1-transporing peptide transportat fixed time-pointsto 60min wasdeterminedby fluorescent naphthalene-2,3-dicarboxaldehyde-derivatized assay(Ex/Em: 405 nm/460 nm. The apparent permeability coefficient(Papp wasused to evaluate the permeability. Expression of PEPT1 protein in Caco-2 cells treated with or without 20 M TF3’G in the presence or absence of inhibitor (10 μM compound C as AMPK inhibitor or 25 μMSTO-609 as CaMKK inhibitor wasevaluated by Western blot.Results:The Pappvalue of Gly-Sar significantly (P<0.05 decreasedin 20 μM purprogallin-treated Caco-2 cellsas well asin TF3’G-treated cells, together with the reduction of PEPT1 expression, while their monomeric catechins did not show any Pappreduction. In TF3’G-treated Caco-2 cells, the recovery of the reduced PEPT1 expression was found by 10 μM compound C,but not STO-609.Conclusion:The study demonstrated that the benzotropolone moiety in theaflavins was a crucial structural requirement for exerting the inhibition of intestinal peptide-transport,and the suppression of PEPT1 expression by theaflavins would be caused by activating LKB1/AMPK pathway

  11. Mast cells modulate transport of CD23/IgE/antigen complex across human intestinal epithelial barrier

    Directory of Open Access Journals (Sweden)

    Ping-Chang Yang

    2009-06-01

    Full Text Available Background: Food allergy and chronic intestinal inflammation are common in western countries. The complex of antigen/IgE is taken up into the body from the gut lumen with the aid of epithelial cell-derived CD23 (low affinity IgE receptor II that plays an important role in the pathogenesis of intestinal allergy. This study aimed to elucidate the role of mast cell on modulation of antigen/IgE complex transport across intestinal epithelial barrier. Methods: Human intestinal epithelial cell line HT29 cell monolayer was used as a study platform. Transepithelial electric resistance (TER and permeability to ovalbumin (OVA were used as the markers of intestinal epithelial barrier function that were recorded in response to the stimulation of mast cell-derived chemical mediators. Results: Conditioned media from naïve mast cell line HMC-1 cells or monocyte cell line THP-1 cells significantly upregulated the expression of CD23 and increased the antigen transport across the epithelium. Treatment with stem cell factor (SCF, nerve growth factor (NGF, retinoic acid (RA or dimethyl sulphoxide (DMSO enhanced CD23 expression in HT29 cells. Conditioned media from SCF, NGF or RA-treated HMC-1 cells, and SCF, NGF, DMSO or RA-treated THP-1 cells enhanced immune complex transport via enhancing the expression of the CD23 in HT29 cells and the release of inflammatory mediator TNF-α. Nuclear factor kappa B inhibitor, tryptase and TNF-α inhibited the increase in CD23 in HT29 cells and prevents the enhancement of epithelial barrier permeability. Conclusions: Mast cells play an important role in modulating the intestinal CD23 expression and the transport of antigen/IgE/CD23 complex across epithelial barrier.

  12. Adsorption of hematite nanoparticles onto Caco-2 cells and the cellular impairments: effect of particle size

    International Nuclear Information System (INIS)

    The increasing applications of engineered nanomaterials nowadays have elevated the potential of human exposure through various routes including inhalation, skin penetration and digestion. To date there is scarce information on a quantitative description of the interactions between nanoparticles (NPs) and cell surfaces and the detrimental effects from the exposure. The purpose of this work was to study in vitro exposure of Caco-2 cells to hematite (α-Fe2O3) NPs and to determine the particle size effects on the adsorption behaviors. Cellular impairment was also investigated and compared. Hematite NPs were synthesized as part of this study with a discrete size distribution and uniform morphology examined by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Caco-2 cells were cultured as a model epithelium to mirror human intestinal cells and used to evaluate the impacts of the exposure to NPs by measuring transepithelial electrical resistance (TEER). Cell surface disruption, localization and translocation of NPs through the cells were analyzed with immunocytochemical staining and confocal microscopy. Results showed that hematite NPs had mean diameters of 26, 53, 76 and 98 nm and were positively charged with minor aggregation in the buffer solution. Adsorption of the four sizes of NPs on cells reached equilibrium within approximately 5 min but adsorption kinetics were found to be size-dependent. The adsorption rates expressed as mg m-2 min-1 were greater for large NPs (76 and 98 nm) than those for small NPs (26 and 53 nm). However, adsorption rates, expressed in units of m-2 min-1, were much greater for small NPs than large ones. After the adsorption equilibrium was reached, the adsorbed mass of NPs on a unit area of cells was calculated and showed no significant size dependence. Longer exposure time (>3 h) induced adverse cellular effects as indicated by the drop in TEER compared to the control cells without the exposure to NPs

  13. Iron-ascorbate-mediated lipid peroxidation causes epigenetic changes in the antioxidant defense in intestinal epithelial cells: impact on inflammation.

    Directory of Open Access Journals (Sweden)

    Sabrina Yara

    Full Text Available INTRODUCTION: The gastrointestinal tract is frequently exposed to noxious stimuli that may cause oxidative stress, inflammation and injury. Intraluminal pro-oxidants from ingested nutrients especially iron salts and ascorbic acid frequently consumed together, can lead to catalytic formation of oxygen-derived free radicals that ultimately overwhelm the cellular antioxidant defense and lead to cell damage. HYPOTHESIS: Since the mechanisms remain sketchy, efforts have been exerted to evaluate the role of epigenetics in modulating components of endogenous enzymatic antioxidants in the intestine. To this end, Caco-2/15 cells were exposed to the iron-ascorbate oxygen radical-generating system. RESULTS: Fe/Asc induced a significant increase in lipid peroxidation as reflected by the elevated formation of malondialdehyde along with the alteration of antioxidant defense as evidenced by raised superoxide dismutase 2 (SOD2 and diminished glutathione peroxidase (GPx activities and genes. Consequently, there was an up-regulation of inflammatory processes illustrated by the activation of NF-κB transcription factor, the higher production of interleukin-6 and cycloxygenase-2 as well as the decrease of IκB. Assessment of promoter's methylation revealed decreased levels for SOD2 and increased degree for GPx2. On the other hand, pre-incubation of Caco-2/15 cells with 5-Aza-2'-deoxycytidine, a demethylating agent, or Trolox antioxidant normalized the activities of SOD2 and GPx, reduced lipid peroxidation and prevented inflammation. CONCLUSION: Redox and inflammatory modifications in response to Fe/Asc -mediated lipid peroxidation may implicate epigenetic methylation.

  14. Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Del Regno, Marisanta; Adesso, Simona; Popolo, Ada [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy); Quaroni, Andrea [Department of Biomedical Sciences, Cornell University, Veterinary Research Tower, Cornell University, Ithaca, NY 14853–6401 (United States); Autore, Giuseppina [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy); Severino, Lorella [Department of Pathology and Animal Health, Division of Toxicology, School of Veterinary Medicine, University of Naples “Federico II”, Via Delpino 1, 80137 Naples (Italy); Marzocco, Stefania, E-mail: smarzocco@unisa.it [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy)

    2015-06-01

    Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. - Highlights: • Nivalenol induces oxidative stress in intestinal epithelial cells (IECs). • Nivalenol increases deoxynivalenol pro-oxidant effects in IECs. • Nivalenol and deoxynivalenol trigger antioxidant response IECs. • These results indicate the importance of mycotoxins co-contamination.

  15. Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells

    International Nuclear Information System (INIS)

    Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. - Highlights: • Nivalenol induces oxidative stress in intestinal epithelial cells (IECs). • Nivalenol increases deoxynivalenol pro-oxidant effects in IECs. • Nivalenol and deoxynivalenol trigger antioxidant response IECs. • These results indicate the importance of mycotoxins co-contamination

  16. Intestinal Epithelial Cell Tyrosine Kinase 2 Transduces IL-22 Signals To Protect from Acute Colitis.

    Science.gov (United States)

    Hainzl, Eva; Stockinger, Silvia; Rauch, Isabella; Heider, Susanne; Berry, David; Lassnig, Caroline; Schwab, Clarissa; Rosebrock, Felix; Milinovich, Gabriel; Schlederer, Michaela; Wagner, Michael; Schleper, Christa; Loy, Alexander; Urich, Tim; Kenner, Lukas; Han, Xiaonan; Decker, Thomas; Strobl, Birgit; Müller, Mathias

    2015-11-15

    In the intestinal tract, IL-22 activates STAT3 to promote intestinal epithelial cell (IEC) homeostasis and tissue healing. The mechanism has remained obscure, but we demonstrate that IL-22 acts via tyrosine kinase 2 (Tyk2), a member of the Jak family. Using a mouse model for colitis, we show that Tyk2 deficiency is associated with an altered composition of the gut microbiota and exacerbates inflammatory bowel disease. Colitic Tyk2(-/-) mice have less p-STAT3 in colon tissue and their IECs proliferate less efficiently. Tyk2-deficient primary IECs show reduced p-STAT3 in response to IL-22 stimulation, and expression of IL-22-STAT3 target genes is reduced in IECs from healthy and colitic Tyk2(-/-) mice. Experiments with conditional Tyk2(-/-) mice reveal that IEC-specific depletion of Tyk2 aggravates colitis. Disease symptoms can be alleviated by administering high doses of rIL-22-Fc, indicating that Tyk2 deficiency can be rescued via the IL-22 receptor complex. The pivotal function of Tyk2 in IL-22-dependent colitis was confirmed in Citrobacter rodentium-induced disease. Thus, Tyk2 protects against acute colitis in part by amplifying inflammation-induced epithelial IL-22 signaling to STAT3. PMID:26432894

  17. Mechanisms of Action of Zinc on Intestinal Epithelial Electrogenic Ion Secretion: Insights into its Anti-Diarrheal Actions

    OpenAIRE

    Bzik, V. A.; Medani, Mekki; Baird, Alan W; Winter, Desmond C.; Brayden, David James

    2012-01-01

    Objectives  Zinc is a useful addition to oral rehydration therapy for acute diarrhoea. We have assessed the mechanism of its epithelial antisecretory action when intestinal epithelial tight junctions were pharmacologically opened. Methods  Rat isolated ileal and colonic mucosae were mounted in Ussing chambers and exposed to ZnSO4 (Zn2+) in the presence of secretagogues and inhibition of short circuit current (Isc) was measured. Key findings  Pre-incubation with basolateral but not api...

  18. Apical Invasion of Intestinal Epithelial Cells by Salmonella typhimurium Requires Villin to Remodel the Brush Border Actin Cytoskeleton

    OpenAIRE

    Lhocine, Nouara; Arena, Ellen T.; Bomme, Perrine; Ubelmann, Florent; Prévost, Marie-Christine; Robine, Sylvie; Sansonetti, Philippe J.

    2015-01-01

    Summary Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, l...

  19. Mn bioavailability by polarized Caco-2 cells: comparison between Mn gluconate and Mn oxyprolinate

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    Fulgenzi Alessandro

    2011-07-01

    Full Text Available Abstract Background Micronutrient inadequate intake is responsible of pathological deficiencies and there is a need of assessing the effectiveness of metal supplementation, frequently proposed to rebalance poor diets. Manganese (Mn is present in many enzymatic intracellular systems crucial for the regulation of cell metabolism, and is contained in commercially available metal supplements. Methods We compared the effects of two different commercial Mn forms, gluconate (MnGluc and oxyprolinate (MnOxP. For this purpose we used the polarized Caco-2 cells cultured on transwell filters, an established in vitro model of intestinal epithelium. Since micronutrient deficiency may accelerate mitochondrial efficiency, the mitochondrial response of these cells, in the presence of MnGluc and MnOxP, by microscopy methods and by ATP luminescence assay was used. Results In the presence of both MnOxP and MnGluc a sustained mitochondrial activity was shown by mitoTraker labeling (indicative of mitochondrial respiration, but ATP intracellular content remained comparable to untreated cells only in the presence of MnOxP. In addition MnOxP transiently up-regulated the antioxidant enzyme Mn superoxide dismutase more efficiently than MnGluc. Both metal treatments preserved NADH and βNADPH diaphorase oxidative activity, avoided mitochondrial dysfunction, as assessed by the absence of a sustained phosphoERK activation, and were able to maintain cell viability. Conclusions Collectively, our data indicate that MnOxP and MnGluc, and primarily the former, produce a moderate and safe modification of Caco-2 cell metabolism, by activating positive enzymatic mechanisms, thus could contribute to long-term maintenance of cell homeostasis.

  20. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells

    OpenAIRE

    Qin, Ying; NAITO, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko

    2011-01-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intest...

  1. Selenium and vitamin E together improve intestinal epithelial barrier function and alleviate oxidative stress in heat-stressed pigs.

    Science.gov (United States)

    Liu, Fan; Cottrell, Jeremy J; Furness, John B; Rivera, Leni R; Kelly, Fletcher W; Wijesiriwardana, Udani; Pustovit, Ruslan V; Fothergill, Linda J; Bravo, David M; Celi, Pietro; Leury, Brian J; Gabler, Nicholas K; Dunshea, Frank R

    2016-07-01

    What is the central question of this study? Oxidative stress may play a role in compromising intestinal epithelial barrier integrity in pigs subjected to heat stress, but it is unknown whether an increase of dietary antioxidants (selenium and vitamin E) could alleviate gut leakiness in heat-stressed pigs. What is the main finding and its importance? Levels of dietary selenium (1.0 p.p.m.) and vitamin E (200 IU kg(-1) ) greater than those usually recommended for pigs reduced intestinal leakiness caused by heat stress. This finding suggests that oxidative stress plays a role in compromising intestinal epithelial barrier integrity in heat-stressed pigs and also provides a nutritional strategy for mitigating these effects. Heat stress compromises the intestinal epithelial barrier integrity of mammals through mechanisms that may include oxidative stress. Our objective was to test whether dietary supplementation with antioxidants, selenium (Se) and vitamin E (VE), protects intestinal epithelial barrier integrity in heat-stressed pigs. Female growing pigs (n = 48) were randomly assigned to four diets containing from 0.2 p.p.m. Se and 17 IU kg(-1) VE (control, National Research Council recommended) to 1.0 p.p.m. Se and 200 IU kg(-1) VE for 14 days. Six pigs from each dietary treatment were then exposed to either thermoneutral (20°C) or heat-stress conditions (35°C 09.00-17.00 h and 28°C overnight) for 2 days. Transepithelial electrical resistance and fluorescein isothiocyanate-dextran (4 kDa; FD4) permeability were measured in isolated jejunum and ileum using Ussing chambers. Rectal temperature, respiratory rate and intestinal HSP70 mRNA abundance increased (all P intestinal barrier function were reduced (P intestinal barrier integrity, associated with a reduction in oxidative stress. PMID:27064134

  2. Probiotic bacteria regulate intestinal epithelial permeability in experimental ileitis by a TNF-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Daniele Corridoni

    Full Text Available We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s of VSL#3-mediated protection on epithelial barrier function and to further investigate the "paradoxical" effects of TNF in preventing SAMP ileitis.Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM, TNF or VSL#3-CM + anti-TNF. Tight junction (TJ proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC from SAMP and control AKR mice.Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice.Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.

  3. Apoptosis in astrovirus-infected CaCo-2 cells

    International Nuclear Information System (INIS)

    Cell death processes during human astrovirus replication in CaCo-2 cells and their underlying mechanisms were investigated. Morphological and biochemical alterations typical of apoptosis were analyzed in infected cells using a combination of techniques, including DAPI staining, the sub-G0/G1 technique and the TUNEL assay. The onset of apoptosis was directly proportional to the virus multiplicity of infection. Transient expression experiments showed a direct link between astrovirus ORF1a encoded proteins and apoptosis induction. A computer analysis of the astrovirus genome revealed the presence of a death domain in the nonstructural protein p38 of unknown function, encoded in ORF1a. Apoptosis inhibition experiments suggested the involvement of caspase 8 in the apoptotic response, and led to a reduction in the infectivity of the virus progeny released to the supernatant. We conclude that apoptotic death of host cells seems necessary for efficient human astrovirus replication and particle maturation

  4. Connexin 26-mediated gap junctional intercellular communication suppresses paracellular permeability of human intestinal epithelial cell monolayers

    International Nuclear Information System (INIS)

    In some cell types, gap junctional intercellular communication (GJIC) is associated with tight junctions. The present study was performed to determine the roles of GJIC in regulation of the barrier function of tight junctions. Caco-2 human colonic cells were used as a monolayer model, and barrier function was monitored by measuring mannitol permeability and transepithelial electrical resistance (TER). The monolayers were chemically disrupted by treatment with oleic acid and taurocholic acid. Western blotting analyses were performed to evaluate the protein levels of connexins, which are components of gap junctional intercellular channels. Cx26 expression was detected in preconfluent Caco-2 cells, and its level increased gradually after the monolayer reached confluency. These results prompted us to examine whether overexpression of Cx26 affects barrier function. Monolayers of Caco-2 cells stably expressing Cx26 showed significantly lower mannitol permeability and higher TER than mock transfectants when the monolayers were chemically disrupted. The levels of claudin-4, an important component of tight junctions, were significantly increased in the stable Cx26 transfectant. These results suggest that Cx26-mediated GJIC may play a crucial role in enhancing the barrier function of Caco-2 cell monolayers

  5. Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine.

    Science.gov (United States)

    Vyas, D; Robertson, C M; Stromberg, P E; Martin, J R; Dunne, W M; Houchen, C W; Barrett, T A; Ayala, A; Perl, M; Buchman, T G; Coopersmith, C M

    2007-06-01

    Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death. PMID:17357092

  6. Chronic low vitamin intake potentiates cisplatin-induced intestinal epithelial cell apoptosis in WNIN rats

    Institute of Scientific and Technical Information of China (English)

    Bodiga Vijayalakshmi; Boindala Sesikeran; Putcha Udaykumar; Subramaniam Kalyanasundaram; Manchala Raghunath

    2006-01-01

    AIM: To investigate if cisplatin alters vitamin status and if VR modulates cisplatin induced intestinal apoptosis and oxidative stress in Wistar/NIN (WNIN) male rats.METHODS: Weanling, WNIN male rats (n = 12 per group) received adlibitum for 17 wk: control diet (20%protein) or the same with 50% vitamin restriction. They were then sub-divided into two groups of six rats each and administered cisplatin (2.61 mg/kg bodyweight)once a week for three wk or PBS (vehicle control).Intestinal epithelial cell (IEC) apoptosis was monitored by morphometry, Annexin-V binding, M30 cytodeath assay and DNA fragmentation. Structural and functional integrity of the villus were assessed by villus height /crypt depth ratio and activities of alkaline phosphatase,lys, ala-dipeptidyl amino-peptidase, respectively. To assess the probable mechanism(s) of altered apoptosis,oxidative stress parameters, caspase-3 activity, and expression of Bcl-2 and Bax were determined.RESULTS: Cisplatin per se decreased plasma vitamin levels and they were the lowest in VR animals treated with cisplatin. As expected VR increased only villus apoptosis, whereas cisplatin increased stem cell apoptosis in the crypt. However, cisplatin treatment of VR rats increased apoptosis both in villus and crypt regions and was associated with higher levels of TBARS,protein carbonyls and caspase-3 activity, but lower GSH concentrations. VR induced decrease in Bcl-2 expression was further lowered by cisplatin. Bax expression,unaffected by VR was increased on cisplatin treatment.Mucosal functional integrity was severely compromised in cisplatin treated VR-rats.CONCLUSION: Low intake of vitamins increases the sensitivity of rats to cisplatin and promotes intestinal epithelial cell apoptosis.

  7. Relationship between β-catenin expression and epithelial cell proliferation in gastric mucosa with intestinal metaplasia

    Institute of Scientific and Technical Information of China (English)

    Adriana Romiti; Pietro Mingazzini; Angelo Zullo; Francesco Borrini; Ida Sarcina; Cesare Hassan; Simon Winn; Silverio Tomao; Aldo Vecchione; Sergio Morini

    2005-01-01

    AIM: To investigate β-catenin expression in patients with intestinal metaplasia, and to look for a possible relationship between β-catenin expression and either epithelial proliferation values or Helicobacter pylori ( H pylori) infection.METHODS: Twenty patients with complete type intestinal metaplasia were studied. β-Catenin expression and epithelial cell proliferation in antral mucosa were assessed using an immunohistochemical analysis. Hpylori infectionwas detected by histology and a rapid urease test.RESULTS: Reduced β-catenin expression on the surface of metaplastic cells was detected in 13 (65%) out of 20 patients. Moreover, in eight (40%) patients intranuclear expression of β-catenin was found. When patients were analyzed according to Hpylori infection, the prevalence of both β-catenin reduction at the cell surface and its intranuclear localization did not significantly differ between infected and uninfected patients. Cell proliferation was higher in patients with intranudear β-catenin expression as compared to the remaining patients, although the difference failed to reach the statistical significance (36±8.9 vs 27.2±11.4, P = 0.06). On the contrary, a similar cell proliferation value was observed between patients with reduced expression of β-catenin on cell surface and those with a normal expression (28.1±11.8 vs26.1±8.8, P= 0.7).Hpyloriinfection significantly increased cell proliferation (33.3±10.2% vs 24.6±7.4%, respectively, P= 0.04).CONCLUSION: Both cell surface reduction and intranuclear accumulation of β-catenin were detected in intestinal metaplasia. The intranuclear localization of β-catenin increases cell proliferation. H pylori infection does not seem to play a direct role in β-catenin alterations, whilst it significantly increases cell proliferation.

  8. Effects of the ionising radiations on the structure and the function of the intestinal epithelial cell

    International Nuclear Information System (INIS)

    The intestinal mucosa is a particularly radio-sensitive tissue and damage may occur following either accidental or therapeutic exposure. the deleterious actions of ionizing radiation are linked to the formation of sometimes overwhelming quantities of reactive oxygen species (R.O.S.). Production of R.O.S. is both direct and indirect from the secondary effects of irradiation. A better comprehension of the underlying mechanisms of injury will lead to more adapted therapeutic approaches to limit the harmful effects of irradiation. The homeostasis of the intestinal epithelium is regulated by three factors: proliferation, apoptosis and differentiation. these three factors were studied using the cell model, HT29, in order to analyze modulations of this balance after irradiation. our results, in agreement with other data, showed the establishment of mitotic delay. This arrest of proliferation was followed by apoptosis to be the major mechanism leading to cell death in this model. thus, for the first time, we have shown that irradiated intestinal epithelial cells preserve their capacity to differentiate. This indicates, although indirectly, that intestinal cells have and preserve an intrinsic capacity restore a functional epithelium. R.O.S. are considered as intermediates between the physical nature of radiations and biological responses. It seems essential to understand anti-oxidant mechanisms used by the cell for defence against the deleterious effects of R.O.S post exposure. This study of several anti-oxidant defence mechanisms of intestinal mucosa, was carried out in vivo in the mouse at different times following abdominal irradiation. We observed an early mitochondrial response in the hours following irradiation revealing this organelle as a particular target. We demonstrated a strong alteration of anti-oxidant capacity as revealed by a decrease in S.O.D.s, catalase and an increase of the G.P.X.s and M.T.s. A part of these modifications appeared to depend on an

  9. Ethanol in utero induces epithelial cell damage and altered kinetics in the developing rat intestine.

    Science.gov (United States)

    Estrada, G; Del Rio, J A; García-Valero, J; López-Tejero, M D

    1996-11-01

    The effect of prenatal ethanol exposure on the intestinal maturation of rat fetuses was investigated to understand the nutritional alterations found in the offspring of alcoholic mothers. Female Wistar rats were maintained on solid diet and 25% ethanol solution as drinking fluid during pregnancy, and non-alcoholic isocaloric pregnant mothers were used as controls. At birth, intestines from unsuckled pups were removed for study. The weight and length of the intestine decreased significantly when ethanol was present in utero. Ultrastructural evaluation of the epithelium revealed loss of contact between neighboring enterocytes and abnormal dilation of the cisternae of the Golgi apparatus in ethanol-exposed pups. Further, increased lysosome-like vesiculation and enhanced lysosomal beta-galactosidase activity was observed in these neonates. The total number of absorptive enterocytes in the epithelium was reduced by 30% in ethanol-exposed neonates as compared to controls, due to altered cell growth and death during fetal life. Ethanol in utero stimulated epithelial cell migration which compensated cell loss, as demonstrated by 5'-Bromodeoxyuridine labeling. These findings could have important implications for the assimilation of nutrients and failure to thrive in infants with fetal alcohol syndrome. PMID:9035346

  10. Small intestinal mucosa expression of putative chaperone fls485

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    Raupach Kerstin

    2010-03-01

    Full Text Available Abstract Background Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. Methods fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. Results fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. Conclusions Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.

  11. Role of myosin light chain kinase in intestinal epithelial barrier defects in a rat model of bowel obstruction

    Directory of Open Access Journals (Sweden)

    Wu Li-Ling

    2010-04-01

    Full Text Available Abstract Background Bowel obstruction is a common cause of abdominal emergency, since the patients are at increased risk of septicemia resulting in high mortality rate. While the compartmentalized changes in enteric microfloral population and augmentation of bacterial translocation (BT have already been reported using experimental obstruction models, alterations in epithelial permeability of the obstructed guts has not been studied in detail. Myosin light chain kinase (MLCK is actively involved in the contraction of epithelial perijunctional actinomyosin ring and thereby increases paracellular permeability. In the current study we attempt to investigate the role of MLCK in epithelial barrier defects using a rat model of simple mechanical obstruction. Methods Wistar rats received intraperitoneal injection of ML-7 (a MLCK inhibitor or vehicle at 24, 12 and 1 hrs before and 12 hrs after intestinal obstruction (IO. The distal small intestine was obstructed with a single ligature placed 10 cm proximal to the ileocecal junction in IO rats for 24 hrs. Sham-operated rats served as controls. Results Mucosal injury, such as villous blunting and increased crypt/villus ratio, was observed in the distal small intestine of IO rats. Despite massive enterocyte shedding, intestinal villi were covered with a contiguous epithelial layer without cell apoptosis. Increased transmural macromolecular flux was noticed in the distal small intestine and the proximal colon after IO. The bacterial colony forming units in the spleen and liver of IO rats were significantly higher than those of sham controls. Addition of ML-7 ameliorated the IO-triggered epithelial MLC phosphorylation, mucosal injury and macromolecular flux, but not the level of BT. Conclusions The results suggest that IO-induced premature enterocytic sloughing and enhanced paracellular antigenic flux were mediated by epithelial MLCK activation. In addition, enteric bacteria may undergo transcytotic routes

  12. MRP2 mediated drug-drug interaction: indomethacin increases sulfasalazine absorption in the small intestine, potentially decreasing its colonic targeting.

    Science.gov (United States)

    Dahan, Arik; Amidon, Gordon L

    2010-02-15

    We have recently shown that efflux transport, mediated by multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP), is responsible for sulfasalazine low-permeability in the small intestine, thereby enabling its colonic targeting and therapeutic action. The purpose of the present study was to evaluate the potential pharmacokinetic interaction between indomethacin and sulfasalazine, in the mechanism of efflux transporter competition. The concentration-dependent effects of indomethacin on sulfasalazine intestinal epithelial transport were investigated across Caco-2 cell monolayers, in both apical to basolateral (AP-BL) and BL-AP directions. The interaction was then investigated in the in situ single-pass rat jejunal perfusion model. Sulfasalazine displayed 30-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. Indomethacin significantly increased AP-BL and decreased BL-AP sulfasalazine Caco-2 transport, in a concentration-dependent manner, with IC(50) values of 75 and 196 microM respectively. In the rat model, higher sulfasalazine concentrations resulted in higher intestinal permeability, consistent with saturation of efflux transporter. Without indomethacin, sulfasalazine demonstrated low rat jejunal permeability (vs. metoprolol). Indomethacin significantly increased sulfasalazine P(eff), effectively shifting it from BCS (biopharmaceutics classification system) Class IV to II. In conclusion, the data indicate that concomitant intake of indomethacin and sulfasalazine may lead to increased absorption of sulfasalazine in the small intestine, thereby reducing its colonic concentration and potentially altering its therapeutic effect. PMID:19944137

  13. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

    OpenAIRE

    Bhargava, Amol; Cotton, James A; Dixon, Brent R.; Gedamu, Lashitew; Robin M. Yates; Buret, Andre G.

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cyste...

  14. Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation

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    McCann Mark J

    2010-12-01

    Full Text Available Abstract Background Intestinal barrier function is important for preserving health, as a compromised barrier allows antigen entry and can induce inflammatory diseases. Probiotic bacteria can play a role in enhancing intestinal barrier function; however, the mechanisms are not fully understood. Existing studies have focused on the ability of probiotics to prevent alterations to tight junctions in disease models, and have been restricted to a few tight junction bridging proteins. No studies have previously investigated the effect of probiotic bacteria on healthy intestinal epithelial cell genes involved in the whole tight junction signalling pathway, including those encoding for bridging, plaque and dual location tight junction proteins. Alteration of tight junction signalling in healthy humans is a potential mechanism that could lead to the strengthening of the intestinal barrier, resulting in limiting the ability of antigens to enter the body and potentially triggering undesirable immune responses. Results The effect of Lactobacillus plantarum MB452 on tight junction integrity was determined by measuring trans-epithelial electrical resistance (TEER across Caco-2 cell layers. L. plantarum MB452 caused a dose-dependent TEER increase across Caco-2 cell monolayers compared to control medium. Gene expression was compared in Caco-2 cells untreated or treated with L. plantarum MB452 for 10 hours. Caco-2 cell RNA was hybridised to human oligonucleotide arrays. Data was analysed using linear models and differently expressed genes were examined using pathway analysis tools. Nineteen tight junction-related genes had altered expression levels in response to L. plantarum MB452 (modified-P 1.2, including those encoding occludin and its associated plaque proteins that anchor it to the cytoskeleton. L. plantarum MB452 also caused changes in tubulin and proteasome gene expression levels which may be linked to intestinal barrier function. Caco-2 tight junctions

  15. Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells

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    Mandayam O. Nandan

    2015-01-01

    Full Text Available Krüppel-like factor 5 (KLF5 is a pro-proliferative transcription factor that is expressed in dividing epithelial cells of the intestinal crypt. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5 has been identified as a stem cell marker in both small intestinal and colonic epithelial cells. To determine whether KLF5 regulates proliferation of intestinal stem cells, we investigated the effects of Klf5 deletion specifically from the intestinal stem cells in adult mice. Mice with inducible intestinal stem cell-specific deletion of Klf5 (Lgr5-Klf5fl/fl were injected with tamoxifen for 5 consecutive days to induce Lgr5-driven Cre expression. Intestinal and colonic tissues were examined by immunohistochemistry at various time points up to 112 days following start of tamoxifen treatment. Klf5 is co-localized in the crypt-based columnar (CBC cells that express Lgr5. By 11 days following the start of tamoxifen treatment, Lgr5-positive crypts from which Klf5 was deleted exhibited a loss of proliferation that was accompanied by an increase in apoptosis. Beginning at 14 days following the start of tamoxifen treatment, both Klf5 expression and proliferation were re-established in the transit-amplifying epithelial cells but not in the Lgr5-positive CBC cells. By 112 days post-treatment, up to 90% of the Lgr5-positive cells from which Klf5 was deleted were lost from the intestinal crypts. These results indicate a critical role for KLF5 in the survival and maintenance of intestinal stem cells.

  16. Loss of Survivin in Intestinal Epithelial Progenitor Cells Leads to Mitotic Catastrophe and Breakdown of Gut Immune Homeostasis.

    Science.gov (United States)

    Martini, Eva; Wittkopf, Nadine; Günther, Claudia; Leppkes, Moritz; Okada, Hitoshi; Watson, Alastair J; Podstawa, Eva; Backert, Ingo; Amann, Kerstin; Neurath, Markus F; Becker, Christoph

    2016-02-01

    A tightly regulated balance of proliferation and cell death of intestinal epithelial cells (IECs) is essential for maintenance of gut homeostasis. Survivin is highly expressed during embryogenesis and in several cancer types, but little is known about its role in adult gut tissue. Here, we show that Survivin is specifically expressed in transit-amplifying cells and Lgr5(+) stem cells. Genetic loss of Survivin in IECs resulted in destruction of intestinal integrity, mucosal inflammation, and death of the animals. Survivin deletion was associated with decreased epithelial proliferation due to defective chromosomal segregation. Moreover, Survivin-deficient animals showed induced phosphorylation of p53 and H2AX and increased levels of cell-intrinsic apoptosis in IECs. Consequently, induced deletion of Survivin in Lgr5(+) stem cells led to cell death. In summary, Survivin is a key regulator of gut tissue integrity by regulating epithelial homeostasis in the stem cell niche. PMID:26832409

  17. Loss of Survivin in Intestinal Epithelial Progenitor Cells Leads to Mitotic Catastrophe and Breakdown of Gut Immune Homeostasis

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    Eva Martini

    2016-02-01

    Full Text Available A tightly regulated balance of proliferation and cell death of intestinal epithelial cells (IECs is essential for maintenance of gut homeostasis. Survivin is highly expressed during embryogenesis and in several cancer types, but little is known about its role in adult gut tissue. Here, we show that Survivin is specifically expressed in transit-amplifying cells and Lgr5+ stem cells. Genetic loss of Survivin in IECs resulted in destruction of intestinal integrity, mucosal inflammation, and death of the animals. Survivin deletion was associated with decreased epithelial proliferation due to defective chromosomal segregation. Moreover, Survivin-deficient animals showed induced phosphorylation of p53 and H2AX and increased levels of cell-intrinsic apoptosis in IECs. Consequently, induced deletion of Survivin in Lgr5+ stem cells led to cell death. In summary, Survivin is a key regulator of gut tissue integrity by regulating epithelial homeostasis in the stem cell niche.

  18. CLMP Is Essential for Intestinal Development, but Does Not Play a Key Role in Cellular Processes Involved in Intestinal Epithelial Development

    OpenAIRE

    Werf, Christine; Hsiao, Nai-hua; Conroy, Siobhan; Paredes, Joana; Ribeiro, Ana; Sribudiani, Yunia; Seruca, Raquel; Hofstra, Robert; Westers, Helga; van IJzendoorn, Sven

    2013-01-01

    textabstractLoss-of-function mutations in CLMP have been found in patients with Congenital Short Bowel Syndrome (CSBS), suggesting that its encoded protein plays a major role in intestinal development. CLMP is a membrane protein that co-localizes with tight junction proteins, but its function is largely unknown. We expressed wild-type (WT)-CLMP and a mutant-CLMP (associated with CSBS) in human intestinal epithelial T84 cells that, as we show here, do not produce endogenous CLMP. We investigat...

  19. Inflammation Controls Sensitivity of Human and Mouse Intestinal Epithelial Cells to Galectin-1.

    Science.gov (United States)

    Muglia, Cecilia I; Papa Gobbi, Rodrigo; Smaldini, Paola; Orsini Delgado, María Lucía; Candia, Martín; Zanuzzi, Carolina; Sambuelli, Alicia; Rocca, Andrés; Toscano, Marta A; Rabinovich, Gabriel A; Docena, Guillermo H

    2016-07-01

    Galectins play key roles in the inflammatory cascade. In this study, we aimed to analyze the effect of galectin-1 (Gal-1) in the function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro-inflammatory cytokines, and Gal-1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal-1 binding and modulation of IECs. We found an enhanced binding of Gal-1 to: (a) murine IECs exposed to IL-1β, TNF, and IL-13; (b) IECs from inflamed areas in intestinal tissue from IBD patients; (c) small bowel of allergic mice; and (d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal-1 favored a tolerogenic micro-environment, whereas high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase-9 and modulation of Bcl-2 protein family members. Our results showed that, when added in the presence of diverse pro-inflammatory cytokines such as tumor necrosis factor (TNF), IL-13 and IL-5, Gal-1 differentially promoted the secretion of growth factors including thymic stromal lymphopoietin (TSLP), epidermal growth factor (EGF), IL-10, IL-25, and transforming growth factor (TGF-β1 ). In conclusion, we found an augmented binding of Gal-1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal-1 concentration. These findings highlight the importance of the inflammatory micro-environment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal-1 and its role in epithelial cell homeostasis. J. Cell. Physiol. 231: 1575-1585, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566180

  20. Thyroid hormone activates Wnt/β-catenin signaling involved in adult epithelial development during intestinal remodeling in Xenopus laevis.

    Science.gov (United States)

    Hasebe, Takashi; Fujimoto, Kenta; Kajita, Mitsuko; Ishizuya-Oka, Atsuko

    2016-08-01

    During amphibian intestinal remodeling, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian epithelium. To clarify molecular mechanisms underlying adult epithelial development, we here focus on TH response genes that are associated with the canonical Wnt pathway. Our quantitative reverse transcription plus polymerase chain reaction and immunohistochemical analyses indicate that all of the genes examined, including β-catenin, c-Myc and secreted frizzle-related protein 2 (SFRP2), are up-regulated in Xenopus laevis intestine during both natural and TH-induced metamorphosis. Moreover, immunoreactivity for nuclear β-catenin becomes detectable in adult stem cells from the start of their appearance and then increases in intensity in adult epithelial primordia derived from the stem cells, which actively proliferate and coexpress Wnt target genes c-Myc and LGR5. These expression profiles strongly suggest the involvement of the canonical Wnt pathway in the maintenance and/or proliferation of adult stem/progenitor cells. More importantly, by using organ cultures of the tadpole intestine, we have experimentally shown that the addition of exogenous SFRP2 protein to the culture medium promotes cell proliferation of the adult epithelial primordia, whereas inhibition of endogenous SFRP2 by its antibody suppresses their proliferation. The inhibition of SFRP2 suppresses larval epithelial changes in shape from simple columnar to stem-cell-like roundish cells, resulting in the failure of epithelial dedifferentiation. Thus, TH-up-regulated SFRP2 in the postembryonic intestine promotes adult stem cell development, possibly by acting as an agonist of both canonical and non-canonical Wnt signaling. PMID:27068920

  1. Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth In Vitro and Pro-Inflammatory Responses in Mouse Experimental Colitis

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    Wang Yu

    2010-11-01

    Full Text Available Abstract Background Serum Amyloid A (SAA is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis. Methods Intestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS colitis was induced in SAA 1/2 double knockout (DKO mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live Escherichia coli. Results Significant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls. Conclusions Intestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..

  2. Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2

    NARCIS (Netherlands)

    Dihal, A.A.; Woutersen, R.A.; Ommen, B.v.; Rietjens, I.M.C.M.; Stierum, R.H.

    2006-01-01

    The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001

  3. E. coli Nissle 1917 Affects Salmonella adhesion to porcine intestinal epithelial cells.

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    Peter Schierack

    Full Text Available BACKGROUND: The probiotic Escherichia coli strain Nissle 1917 (EcN has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. CONCLUSIONS: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.

  4. In vivo gene expression profiling of human intestinal epithelial cells: analysis by laser microdissection of formalin fixed tissues

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    Sabir Sadiah

    2008-05-01

    Full Text Available Abstract Background The small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However, the differences in transcriptional programming and the molecular machinery that governs the migration, adhesion, and differentiation of intestinal epithelial cell lineages in humans remain under-explored. To increase our understanding of these mechanisms, we have evaluated gene expression patterns of ileal epithelial cells isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of healthy human small intestinal mucosa. Expression profiles in villus and crypt epithelium were determined by DNA microarray, quantitative real-time PCR, and immunohistochemistry based methods. The expression levels of selected epithelial biomarkers were also compared between gastrointestinal tissues. Results Previously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations (GEO accession: GSE10629. Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5 and regenerating islet-derived 3 alpha (Reg3A, were detected exclusively within the small bowel crypt, most notably in the ileum in comparison to other sites along the gastrointestinal tract. Conclusion The elucidation of differential gene expression patterns between crypt and villus epithelial cell lineages in human ileal tissue provides novel insights into the molecular machinery that mediates their functions and spatial organization. Moreover, our findings establish an important framework of knowledge for future investigations of human gastrointestinal diseases.

  5. The insulin receptor substrate 1 (irs1) in intestinal epithelial differentiation and in colorectal cancer.

    OpenAIRE

    Esposito, DL; Aru, F; Lattanzio, R; Morgano, A.; Abbondanza, M; Malekzadeh, R; BISHEHSARI F; Valanzano, R; Russo, A; Piantelli, M; Moschetta, A; Lotti, LV; MARIANI-COSTANTINI R

    2012-01-01

    Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and ...

  6. Processing of whey modulates proliferative and immune functions in intestinal epithelial cells.

    Science.gov (United States)

    Nguyen, Duc Ninh; Sangild, Per T; Li, Yanqi; Bering, Stine B; Chatterton, Dereck E W

    2016-02-01

    Whey protein concentrate (WPC) is often subjected to heat treatment during industrial processing, resulting in protein denaturation and loss of protein bioactivity. We hypothesized that WPC samples subjected to different degrees of thermal processing are associated with different levels 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 of whey, with the lowest level being found in WPC derived from acid whey. Following acid activation, WPC from acid whey enhanced IEC proliferation compared with WPC from sweet whey or nonactivated WPC. Low-heat-treated WPC from acid whey induced greater secretion of IL-8 in IEC than either standard WPC from acid whey or low-heat-treated WPC from sweet whey. Following acid activation (to activate growth factors), low-heat-treated WPC from sweet whey induced higher IL-8 levels in IEC compared with standard WPC from sweet whey. In conclusion, higher levels of bioactive proteins in low-heat-treated WPC, especially from acid whey, may enhance proliferation and cytokine responses of IEC. These considerations could be important to maintain optimal bioactivity of infant formulas, including their maturational and immunological effects on the developing intestine. PMID:26709184

  7. Comparative Study of Domoic Acid and Okadaic Acid Induced - Chromosomal Abnormalities in the CACO-2 Cell Line

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    Edmond E. Creppy

    2006-03-01

    Full Text Available Okadaic Acid (OA the major diarrheic shellfish poisoning (DSP toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA, the major Amnesic Shellfish Poisoning (ASP toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA and domoic acid (DA to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.

  8. Ethanolamine enhances the proliferation of intestinal epithelial cells via the mTOR signaling pathway and mitochondrial function.

    Science.gov (United States)

    Yang, Huansheng; Xiong, Xia; Li, Tiejun; Yin, Yulong

    2016-05-01

    Ethanolamine (Etn), which is the base constituent of phosphatidylethanolamine, a major phospholipid in animal cell membranes, is required for the proliferation of many types of mammalian epithelial cells. However, it is not clear whether the proliferation of intestinal epithelial cells requires Etn. The present study was conducted to examine the effects of Etn on the proliferation of intestinal epithelial cells and to elucidate the underlying mechanisms. The addition of Etn at 100 or 200 μM was found to enhance the proliferation of IPEC-1 cells. The expression of cell cycle-related proteins CDK4, RB3, cyclin A, and PCNA was also enhanced by Etn. Moreover, the expression or phosphorylation levels of the mammalian target of rapamycin (mTOR) signaling pathway protein and the expression of proteins related to mitochondrial function were also affected by Etn in IPEC-1 cells. These results indicate that Etn promotes the proliferation of intestinal epithelial cells by exerting effects on mTOR signaling pathway and mitochondrial function. PMID:27083163

  9. Molecular & Biochemical Parasitology Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells

    Science.gov (United States)

    Ringqvist, Emma; Palm, J.E. Daniel; Skarin, Hanna; Hehl, Adrian B.; Weiland, Malin; Davids, Barbara J.; Reiner, David S.; Griffiths, William J.; Eckmann, Lars; Gillin, Frances D.; Svärd, Staffan G.

    2012-01-01

    Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell–cell interactions, since only small amounts of argi-nine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine. PMID:18359106

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

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    Wang YB

    2013-10-01

    Full Text Available Yanbo Wang, Xuxia Yan, Linglin Fu Marine Resources and Nutrition Biology Research Center, Food Quality and Safety Department, Zhejiang Gongshang University, Hangzhou, People's Republic of China Abstract: Nano-selenium (Se, with its high bioavailability and low toxicity, has attracted wide attention for its potential application in the prevention of oxidative damage in animal tissues. However, the effect of nano-Se of different sizes on the intestinal epithelial cells of the crucian carp (Carassius auratus gibelio is poorly understood. Our study showed that different sizes and doses of nano-Se have varied effects on the cellular protein contents and the enzyme activities of secreted lactate dehydrogenase, intracellular sodium potassium adenosine triphosphatase, glutathione peroxidase, and superoxide dismutase. It was also indicated that nano-Se had a size-dependent effect on the primary intestinal epithelial cells of the crucian carp. Thus, these findings may bring us a step closer to understanding the size effect and the bioavailability of nano-Se on the intestinal tract of the crucian carp. Keywords: selenium nanoparticle, intestinal epithelial cell, crucian carp, primary culture

  11. Pathway and single gene analyses of inhibited Caco-2 differentiation by ascorbate-stabilized quercetin suggest enhancement of cellular processes associated with development of colon cancer.

    Science.gov (United States)

    Dihal, Ashwin A; Tilburgs, Chantal; van Erk, Marjan J; Rietjens, Ivonne M C M; Woutersen, Ruud A; Stierum, Rob H

    2007-08-01

    The aim was to investigate mechanisms contributing to quercetin's previously described effects on cell-proliferation and -differentiation, which contradicted its proposed anticarcinogenic potency. In a 10-day experiment, 40 microM quercetin stabilized by 1 mM ascorbate reduced Caco-2 differentiation up to 50% (p < 0.001). Caco-2 RNA from days 5 and 10, hybridized on HG-U133A2.0 Affymetrix GeneChips(R), showed 1,743 affected genes on both days (p < 0.01). All 14 Caco-2 differentiation-associated genes showed decreased expression (p < 0.01), including intestinal alkaline phosphatase, that was confirmed technically (qRT-PCR) and functionally (enzyme-activity). The 1,743 genes contributed to 27 pathways (p < 0.05) categorized under six gene ontology (GO) processes, including apoptosis and cell-cycle. Genes within these GO-processes showed fold changes that suggest increased cell-survival and -proliferation. Furthermore, quercetin down-regulated expression of genes involved in tumor-suppression and phase II metabolism, and up-regulated oncogenes. Gene expression changes mediated by ascorbate-stabilized quercetin were concordant with those occurring in human colorectal carcinogenesis ( approximately 80-90%), but were opposite to those previously described for Caco-2 cells exposed to quercetin without ascorbate ( approximately 75-90%). In conclusion, gene expression among Caco-2 cells exposed to ascorbate-stabilized quercetin showed mechanisms contrary to what is expected for a cancer-preventive agent. Whether this unexpected in vitro effect is relevant in vivo, remains to be elucidated. PMID:17639512

  12. The effect of milk components and storage conditions on the virulence of Listeria monocytogenes as determined by a Caco-2 cell assay.

    Science.gov (United States)

    Pricope-Ciolacu, Luminita; Nicolau, Anca Ioana; Wagner, Martin; Rychli, Kathrin

    2013-08-16

    Nearly all cases of human listeriosis have been associated with consumption of contaminated food, therefore the investigation of the virulence of Listeria (L.) monocytogenes after exposure to environmental conditions in food matrices is critical in order to understand and control its impact on public health. As milk and dairy products have been implicated in more than half of the listeriosis outbreaks, we investigated the in vitro virulence of L. monocytogenes incubated in different milk types at various storage conditions. Incubation in pasteurized milk at refrigeration conditions (4°C) revealed a higher invasion and intracellular proliferation of four different L. monocytogenes strains compared to raw milk using human intestinal epithelial Caco-2 cells. Furthermore the period of storage, which increased L. monocytogenes cell numbers, decreased in vitro virulence. However, L. monocytogenes stored for 3weeks at 4°C in milk are still able to invade and proliferate into the host cell. Interestingly abused storage temperatures (25°C and 30°C) for a short time period (2h) revealed an attenuated impact on the in vitro virulence of L. monocytogenes compared to the storage temperature of 4°C. Regarding the major milk compounds, the level of milk fat significantly affected the in vitro virulence of L. monocytogenes. Pre-incubation in milk with high fat content (3.6%) resulted in a lower invasion capability compared to milk with low fat content. In contrast casein and lactose did not influence the invasiveness of L. monocytogenes into the host cell. In conclusion our study shows that the milk environment and different storage conditions influence the in vitro virulence of L. monocytogenes, both of which have to be considered in the risk assessment of contaminated food. PMID:23831732

  13. Protective Effects of Ferulic Acid against Heat Stress-Induced Intestinal Epithelial Barrier Dysfunction In Vitro and In Vivo.

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    Shasha He

    Full Text Available Heat stress is important in the pathogenesis of intestinal epithelial barrier dysfunction. Ferulic acid (FA, a phenolic acid widely found in fruits and vegetables, can scavenge free radicals and activate cell stress responses. This study is aimed at investigating protective effects of FA on heat stress-induced dysfunction of the intestinal epithelial barrier in vitro and in vivo. Intestinal epithelial (IEC-6 cells were pretreated with FA for 4 h and then exposed to heat stress. Heat stress caused decreased transepithelial electrical resistance (TER and increased permeability to 4-kDa fluorescein isothiocyanate (FITC-dextran (FD4. Both effects were inhibited by FA in a dose-dependent manner. FA significantly attenuated the decrease in occludin, ZO-1 and E-cadherin expression observed with heat stress. The distortion and redistribution of occludin, ZO-1 and E-cadherin proteins were also effectively prevented by FA pretreatment. Moreover, heat stress diminished electron-dense material detected in tight junctions (TJs, an effect also alleviated by FA in a dose-dependent manner. In an in vivo heat stress model, FA (50 mg/kg was administered to male Sprague-Dawley rats for 7 consecutive days prior to exposure to heat stress. FA pretreatment significantly attenuated the effects of heat stress on the small intestine, including the increased FD4 permeability, disrupted tight junctions and microvilli structure, and reduced occludin, ZO-1 and E-cadherin expression. Taken together, our results demonstrate that FA pretreatment is potentially protective against heat stress-induced intestinal epithelial barrier dysfunction.

  14. HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

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    Giuliana Mastropietro

    2015-01-01

    Full Text Available The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.

  15. The expression of apoB mRNA editing factors is not the sole determinant for the induction of editing in differentiating Caco-2 cells

    International Nuclear Information System (INIS)

    Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is ∼80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expression of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.

  16. 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 < 0.05) than the strains isolated from...... meat and food-processing environments (3008, 3126, and 4140) after grown in BHI at 30 degrees C. This attenuation could not be ascribed to a defective Internalin A as all strains encoded an intact inlA gene. To determine the influence of food products on virulence, the ability of L. monocytogenes to...

  17. Cellular inhibitor of apoptosis protein 2 controls human colonic epithelial restitution, migration, and Rac1 activation

    DEFF Research Database (Denmark)

    Seidelin, Jakob Benedict; Larsen, Sylvester; Linnemann, Dorte;

    2015-01-01

    study was to investigate the role of cIAP2 for wound healing in the normal human colon. Wound tissue was generated by taking rectosigmoidal biopsies across an experimental ulcer in healthy subjects after 5, 24, and 48 h. In experimental ulcers, the expression of cIAP2 in regenerating intestinal...... epithelial cells (IECs) was increased at the wound edge after 24 h (P < 0.05), returned to normal after reepithelialization, and correlated with the inflammatory reaction in the experimental wounds (P < 0.001). cIAP2 was induced in vitro in regenerating Caco2 IECs after wound infliction (P < 0.01). Knockdown...

  18. Commenting on the effects of surface treated- and non-surface treated TiO2 in the Caco-2 cell model

    Directory of Open Access Journals (Sweden)

    Faust James J

    2012-11-01

    Full Text Available Abstract In a recent work published in Particle and Fibre Toxicology by Fisichella and coworkers investigating surface-modified TiO2 nanoparticle exposure in a model human intestinal epithelium (Caco-2, albeit degraded to mimic conditions in the gut and exposure to natural sunlight, purportedly resulted in no toxic effects. The authors (Fisichella et al. claim to have confirmed the results of a 2010 report by Koeneman et al. However, the study by Koeneman and colleagues revealed significant effects of unmodified TiO2 nanoparticles. These contradicting data warrant further investigation into the possible effects of aluminum hydroxide, as these nanoparticles appear to have resulted in an abnormal apical surface in Caco-2 cells.

  19. Bacteria regulate intestinal epithelial cell differentiation factors both in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Svetlana Becker

    Full Text Available BACKGROUND: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. METHODS: Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted. RESULTS: Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice. CONCLUSIONS: Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.

  20. Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Junqiang; Doi, Hiroshi [Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, California (United States); Saar, Matthias; Santos, Jennifer [Department of Urology, School of Medicine, Stanford University, Stanford, California (United States); Li, Xuejun; Peehl, Donna M. [Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, California (United States); Knox, Susan J., E-mail: sknox@stanford.edu [Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, California (United States)

    2013-12-01

    Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome. The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.

  1. Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

    International Nuclear Information System (INIS)

    Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome. The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy

  2. Strategies of the protozoan parasite Entamoeba histolytica to evade the innate immune responses of intestinal epithelial cells

    Indian Academy of Sciences (India)

    S Ankri

    2002-11-01

    Molecules expressed by the pathogenic ameoba Entamoeba histolytica but weakly expressed or absent from the non-pathogenic ameoba Entamoeba dispar could be used by intestinal epithelial cells to discriminate between the two species and to initiate an appropriate inflammatory response. Among the possible molecules involved in this identification are the Gal/GalNac lectin and the lipophosphoglycan. Once the inflammatory response is initiated, E. histolytica trophozoites have to protect themselves against reactive nitrogen intermediates produced by intestinal epithelial cells, oxygen intermediates, and cytotoxic molecules released by activated neutrophils. By screening the E. histolytica genome, we have identified proteins that may play a role in the defence strategy of the parasite. One of these proteins, a serine proteinase inhibitor, inhibits human neutrophil cathepsin G, a key component of the host defence.

  3. Short chain fatty acids stimulate epithelial mucin 2 expression through differential effects on prostaglandin E(1) and E(2) production by intestinal myofibroblasts

    OpenAIRE

    Willemsen, L.E.; Koetsier, M A; Deventer, van, S.J.H.; Tol, van, M.J.

    2003-01-01

    Background: The mucus layer protects the gastrointestinal mucosa from mechanical, chemical, and microbial challenge. Mucin 2 (MUC-2) is the most prominent mucin secreted by intestinal epithelial cells. There is accumulating evidence that subepithelial myofibroblasts regulate intestinal epithelial cell function and are an important source of prostaglandins (PG). PG enhance mucin secretion and are key players in mucoprotection. The role of bacterial fermentation products in these processes dese...

  4. Inhibition of the NF-kappaB pathway in human intestinal epithelial cells by commensal Streptococcus salivarius.

    Science.gov (United States)

    Kaci, Ghalia; Lakhdari, Omar; Doré, Joël; Ehrlich, S Dusko; Renault, Pierre; Blottière, Hervé M; Delorme, Christine

    2011-07-01

    Streptococcus salivarius exhibited an anti-inflammatory effect on intestinal epithelial cells (IECs) and monocytes. Strains were screened using a reporter clone, HT-29/kB-luc-E, induced by tumor necrosis factor alpha (TNF-α). Supernatant from each strain downregulated NF-κB activation. The two most efficient strains produced an active metabolite (<3 kDa) which was able to downregulate the secretion of the proinflammatory chemokine interleukin-8 (IL-8). PMID:21602373

  5. Inhibition of the NF-κB Pathway in Human Intestinal Epithelial Cells by Commensal Streptococcus salivarius ▿ †

    Science.gov (United States)

    Kaci, Ghalia; Lakhdari, Omar; Doré, Joël; Ehrlich, S. Dusko; Renault, Pierre; Blottière, Hervé M.; Delorme, Christine

    2011-01-01

    Streptococcus salivarius exhibited an anti-inflammatory effect on intestinal epithelial cells (IECs) and monocytes. Strains were screened using a reporter clone, HT-29/kB-luc-E, induced by tumor necrosis factor alpha (TNF-α). Supernatant from each strain downregulated NF-κB activation. The two most efficient strains produced an active metabolite (<3 kDa) which was able to downregulate the secretion of the proinflammatory chemokine interleukin-8 (IL-8). PMID:21602373

  6. Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

    OpenAIRE

    Tomosada, Yohsuke; Villena, Julio; Murata, Kozue; Chiba, Eriko; Shimazu, Tomoyuki; Aso, Hisashi; Iwabuchi, Noriyuki; Xiao, Jin-zhong; Saito, Tadao; KITAZAWA, Haruki

    2013-01-01

    Background We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains. Objective The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflamma...

  7. Identification of Store-independent and Store-operated Ca2+ Conductances in Caenorhabditis elegans Intestinal Epithelial Cells

    OpenAIRE

    Estevez, Ana Y.; Roberts, Randolph K.; Strange, Kevin

    2003-01-01

    The nematode Caenorhabditis elegans offers significant experimental advantages for defining the genetic basis of diverse biological processes. Genetic and physiological analyses have demonstrated that inositol-1,4,5-trisphosphate (IP3)–dependent Ca2+ oscillations in intestinal epithelial cells play a central role in regulating the nematode defecation cycle, an ultradian rhythm with a periodicity of 45–50 s. Patch clamp studies combined with behavioral assays and forward and reverse genetic sc...

  8. Probiotic Escherichia coli Nissle 1917 and commensal E. coli K12 differentially affect the inflammasome in intestinal epithelial cells

    OpenAIRE

    Becker, Helen M; Apladas, Aretussa; Scharl, Michael; Fried, Michael; Rogler, Gerhard

    2014-01-01

    BACKGROUND: The probiotic bacterial strain Escherichia coli Nissle 1917 (EcN) is used for the treatment of ulcerative colitis (UC), diarrhea and constipation. Its beneficial effects in the treatment of UC have been demonstrated in several controlled clinical studies; however, the mechanism of action on the cellular level is still not completely clear. The intracellular pattern recognition receptor NLRP3 is expressed in intestinal epithelial cells (IEC), activates caspase-1 within the inflamma...

  9. Lactobacillus acidophilus induces cytokine and chemokine production via NF-κB and p38 mitogen-activated protein kinase signaling pathways in intestinal epithelial cells.

    Science.gov (United States)

    Jiang, Yujun; Lü, Xuena; Man, Chaoxin; Han, Linlin; Shan, Yi; Qu, Xingguang; Liu, Ying; Yang, Shiqin; Xue, Yuqing; Zhang, Yinghua

    2012-04-01

    Intestinal epithelial cells can respond to certain bacteria by producing an array of cytokines and chemokines which are associated with host immune responses. Lactobacillus acidophilus NCFM is a characterized probiotic, originally isolated from human feces. This study aimed to test the ability of L. acidophilus NCFM to stimulate cytokine and chemokine production in intestinal epithelial cells and to elucidate the mechanisms involved in their upregulation. In experiments using intestinal epithelial cell lines and mouse models, we observed that L. acidophilus NCFM could rapidly but transiently upregulate a number of effector genes encoding cytokines and chemokines such as interleukin 1α (IL-1α), IL-1β, CCL2, and CCL20 and that cytokines showed lower expression levels with L. acidophilus NCFM treatment than chemokines. Moreover, L. acidophilus NCFM could activate a pathogen-associated molecular pattern receptor, Toll-like receptor 2 (TLR2), in intestinal epithelial cell lines. The phosphorylation of NF-κB p65 and p38 mitogen-activated protein kinase (MAPK) in intestinal epithelial cell lines was also enhanced by L. acidophilus NCFM. Furthermore, inhibitors of NF-κB (pyrrolidine dithiocarbamate [PDTC]) and p38 MAPK (SB203580) significantly reduced cytokine and chemokine production in the intestinal epithelial cell lines stimulated by L. acidophilus NCFM, suggesting that both NF-κB and p38 MAPK signaling pathways were important for the production of cytokines and chemokines induced by L. acidophilus NCFM. PMID:22357649

  10. Short-Chain Fatty Acids Regulate Secretion of IL-8 from Human Intestinal Epithelial Cell Lines in vitro.

    Science.gov (United States)

    Asarat, M; Vasiljevic, T; Apostolopoulos, V; Donkor, O

    2015-01-01

    Short-chain fatty acids (SCFAs) including acetate, propionate and butyrate play an important role in the physiological functions of epithelial cells and colonocytes, such as immune response regulation. Human intestinal epithelial cells (IECs) contribute in intestinal immune response via different ways, such as production of different immune factors including Interleukin (IL) IL-8, which act as chemoattractant for neutrophils, and subsequently enhance inflammation. Therefore, we aimed to evaluate the effects of SCFAs on IECs viability and production of IL-8 in vitro. SCFAs were co-cultured with either normal intestinal epithelial (T4056) or adenocarcinoma derived (HT-29) cell lines for 24-96 h in the presence of E.coli lipopolysaccharides (LPS). Cell viability, proliferation, production of IL-8 and expression of IL-8 mRNA were determined in the cell cultures. The result showed that 20 mM of SCFAs was non-cytotoxic to T4056 and enhanced their growth, whereas the growth of HT-29 was inhibited. The SCFAs down regulated LPS-stimulated IL-8 secretion with different response patterns, but no obvious effects on the release of IL-8 from non LPS- stimulated cells. In conclusion, SCFAs showed regulatory effect on release of LPS-stimulated IL-8 as well as the expression of mRNA of IL-8; these might explain the anti-inflammatory and anti-carcinogenic mechanism of SCFAs. PMID:26436853

  11. Neonatal Fc Receptor-Mediated IgG Transport Across Porcine Intestinal Epithelial Cells: Potentially Provide the Mucosal Protection.

    Science.gov (United States)

    Guo, Jinyue; Li, Fei; He, Qigai; Jin, Hui; Liu, Mei; Li, Shaowen; Hu, Sishun; Xiao, Yuncai; Bi, Dingren; Li, Zili

    2016-06-01

    It has been well characterized that piglets can absorb colostrum IgG across the intestine to neonatal bloodstream and a certain level of IgG has been found in the mucosal secretions of the porcine intestinal tract. However, little is known about how the maternal IgG transport across the intestinal barrier and how IgG enter the lumen of intestinal tract. In this study, we demonstrated that the porcine neonatal Fc receptor (pFcRn) was expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2) as well as in kidney cells (PK-15), and pFcRn was mainly distributed in the apical side of the polarized IPEC-J2 cells. Analyzing the phylogenetic relatedness of this gene we found that swine and human neonatal Fc receptor (FcRn) amino acid sequence are closer than rodents. We also showed that pFcRn mediated bidirectional IgG transport across polarized IPEC-J2 cells and bound to IgG in a pH-dependent manner. Furthermore, pFcRn-transcytosed viral-specific IgG reduced the transmissible gastroenteritis virus (TGEV) yield from the luminal direction by a 50% tissue culture infective dose (TCID50) assay. Our results indicate that pFcRn-dependent bidirectional IgG transport across the intestinal epithelium plays critical role in the acquisition of humoral immunity in early life and in host defense at mucosal surfaces. PMID:26982157

  12. A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

    Science.gov (United States)

    Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

  13. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yanlong [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Chunhong [Second Hospital, Jilin University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Yuhua [College of Food Science and Engineering, Jilin Agricultural University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Ma, Zhenhua [First Hospital, Xi' an Jiaotong University, Xi' an (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); McClain, Craig [Department of Medicine, University of Louisville, Louisville, KY (United States); Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States); Robley Rex Veterans Affairs Medical Center, Louisville, KY (United States); Li, Xiaokun [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Feng, Wenke, E-mail: wenke.feng@louisville.edu [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States)

    2012-10-15

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

  14. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    International Nuclear Information System (INIS)

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl2), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl2 treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl2 administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl2-induced reactive oxygen species (ROS) formation and completely negated CoCl2-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl2 administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl2 increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl2-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and absorption. -- Graphical abstract: Physical and

  15. Hypocholesterolaemic Activity of Lupin Peptides: Investigation on the Crosstalk between Human Enterocytes and Hepatocytes Using a Co-Culture System Including Caco-2 and HepG2 Cells.

    Science.gov (United States)

    Lammi, Carmen; Zanoni, Chiara; Ferruzza, Simonetta; Ranaldi, Giulia; Sambuy, Yula; Arnoldi, Anna

    2016-01-01

    Literature indicates that peptic and tryptic peptides derived from the enzymatic hydrolysis of lupin protein are able to modulate cholesterol metabolism in human hepatic HepG2 cells and that part of these peptides are absorbed in a small intestine model based on differentiated human Caco-2 cells. In this paper, a co-culture system, including Caco-2 and HepG2 cells, was investigated with two objectives: (a) to verify whether cholesterol metabolism in HepG2 cells was modified by the peptides absorption through Caco-2 cells; (b) to investigate how lupin peptides influence cholesterol metabolism in Caco-2 cells. The experiments showed that the absorbed peptides, not only maintained their bioactivity on HepG2 cells, but that this activity was improved by the crosstalk of the two cells systems in co-culture. In addition, lupin peptides showed a positive influence on cholesterol metabolism in Caco-2 cells, decreasing the proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion. PMID:27455315

  16. Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

    Directory of Open Access Journals (Sweden)

    Duan Rui-Dong

    2010-04-01

    Full Text Available Abstract Background Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1 protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells. Methods Caco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification. Results We found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 μM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 μM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively. Conclusion

  17. Interferon-γ induces expression of MHC class II on intestinal epithelial cells and protects mice from colitis.

    Directory of Open Access Journals (Sweden)

    Christoph Thelemann

    Full Text Available Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD and involve CD4(+ T cells, which are activated by major histocompatibility complex class II (MHCII molecules on antigen-presenting cells (APCs. However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC affects CD4(+ T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL-10 receptor-blocking antibodies (anti-IL10R mAb. To assess the role of interferon (IFN-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+ T-helper type (Th1 cells - but not group 3 innate lymphoid cells (ILCs or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+ T cells and forkhead box P3 (FoxP3(+ regulatory T (Treg cells. IFN-γ produced mainly by CD4(+ T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

  18. Altered intestinal microbial flora and impaired epithelial barrier structure and function in CKD: the nature, mechanisms, consequences and potential treatment.

    Science.gov (United States)

    Vaziri, Nosratola D; Zhao, Ying-Yong; Pahl, Madeleine V

    2016-05-01

    Chronic kidney disease (CKD) results in systemic inflammation and oxidative stress which play a central role in CKD progression and its adverse consequences. Although many of the causes and consequences of oxidative stress and inflammation in CKD have been extensively explored, little attention had been paid to the intestine and its microbial flora as a potential source of these problems. Our recent studies have revealed significant disruption of the colonic, ileal, jejunal and gastric epithelial tight junction in different models of CKD in rats. Moreover, the disruption of the epithelial barrier structure and function found in uremic animals was replicated in cultured human colonocytes exposed to uremic human plasma in vitro We have further found significant changes in the composition and function of colonic bacterial flora in humans and animals with advanced CKD. Together, uremia-induced impairment of the intestinal epithelial barrier structure and function and changes in composition of the gut microbiome contribute to the systemic inflammation and uremic toxicity by accommodating the translocation of endotoxin, microbial fragments and other noxious luminal products in the circulation. In addition, colonic bacteria are the main source of several well-known pro-inflammatory uremic toxins such as indoxyl sulfate, p-cresol sulfate, trimethylamine-N-oxide and many as-yet unidentified retained compounds in end-stage renal disease patients. This review is intended to provide an overview of the effects of CKD on the gut microbiome and intestinal epithelial barrier structure and their role in the pathogenesis of systemic inflammation and uremic toxicity. In addition, potential interventions aimed at mitigating these abnormalities are briefly discussed. PMID:25883197

  19. Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics.

    Science.gov (United States)

    Pastorelli, Luca; De Salvo, Carlo; Mercado, Joseph R; Vecchi, Maurizio; Pizarro, Theresa T

    2013-01-01

    The gut mucosa is constantly challenged by a bombardment of foreign antigens and environmental microorganisms. As such, the precise regulation of the intestinal barrier allows the maintenance of mucosal immune homeostasis and prevents the onset of uncontrolled inflammation. In support of this concept, emerging evidence points to defects in components of the epithelial barrier as etiologic factors in the pathogenesis of inflammatory bowel diseases (IBDs). In fact, the integrity of the intestinal barrier relies on different elements, including robust innate immune responses, epithelial paracellular permeability, epithelial cell integrity, as well as the production of mucus. The purpose of this review is to systematically evaluate how alterations in the aforementioned epithelial components can lead to the disruption of intestinal immune homeostasis, and subsequent inflammation. In this regard, the wealth of data from mouse models of intestinal inflammation and human genetics are pivotal in understanding pathogenic pathways, for example, that are initiated from the specific loss of function of a single protein leading to the onset of intestinal disease. On the other hand, several recently proposed therapeutic approaches to treat human IBD are targeted at enhancing different elements of gut barrier function, further supporting a primary role of the epithelium in the pathogenesis of chronic intestinal inflammation and emphasizing the importance of maintaining a healthy and effective intestinal barrier. PMID:24062746

  20. Central role of the gut epithelial barrier in pathogenesis of chronic intestinal inflammation: Lessons learned from animal models and human genetics

    Directory of Open Access Journals (Sweden)

    Luca ePastorelli

    2013-09-01

    Full Text Available The gut mucosa is constantly challenged by a bombardment of foreign antigens and environmental microorganisms. As such, the precise regulation of the intestinal barrier allows the maintenance of mucosal immune homeostasis and prevents the onset of uncontrolled inflammation. In support of this concept, emerging evidence points to defects in components of the epithelial barrier as etiologic factors in the pathogenesis of inflammatory bowel diseases (IBDs. In fact, the integrity of the intestinal barrier relies on different elements, including robust innate immune responses, epithelial paracellular permeability, epithelial cell integrity, as well as the production of mucus. The purpose of this review is to systematically evaluate how alterations in the aforementioned epithelial components can lead to the disruption of intestinal immune homeostasis, and subsequent inflammation. In this regard, the wealth of data from mouse models of intestinal inflammation and human genetics are pivotal in understanding pathogenic pathways, for example, that are initiated from the specific loss of function of a single protein leading to the onset of intestinal disease. On the other hand, several recently proposed therapeutic approaches to treat human IBD are targeted at enhancing different elements of gut barrier function, further supporting a primary role of the epithelium in the pathogenesis of chronic intestinal inflammation and emphasizing the importance of maintaining a healthy and effective intestinal barrier.

  1. Effects of biodegradable Mg–6Zn alloy extracts on apoptosis of intestinal epithelial cells

    International Nuclear Information System (INIS)

    Highlights: ► We evaluated the effects of Mg–6Zn alloys on apoptosis of IEC-6 cells. ► The apoptosis was evaluated by investigating the expression of caspase-1 and Bcl-2. ► The IEC-6 cells displayed better cell functions in 60% or 20% extract. ► The conspicuous alkaline environment is disadvantageous to apoptosis of IEC cells. ► The excessive Mg concentration is disadvantageous to apoptosis of IEC-6 cells. - Abstract: In this study, intestinal epithelial cells (IEC)-6 were cultured in different concentration extracts of Mg–6Zn alloys for different time periods. To achieve a total of three concentrations (100%, 60% and 20% concentration), the extracts were serially diluted with Dulbecco's modified Eagle medium High Glucose to observe a dose–response relationship. We studied the indirect effects of Mg–6Zn alloys on IEC-6 cells apoptosis. The apoptosis of IEC-6 cells was measured using flow cytometry. And the apoptosis of IEC-6 cells was evaluated by investigating the expression of caspase-1and Bcl-2 using real-time polymerase chain reaction (PCR) and Western blotting tests. It was found that the levels of apoptosis in IEC-6 cells cultured in 100% Mg–6Zn alloy extracts were significantly higher than those in 60% and 20% extracts; the 100% extract can down-regulate expression of Bcl-2 after culture. The in vitro results indicated that the conspicuous alkaline environment and excessive Mg concentration, even Zn concentration caused by rapid corrosion of Mg–6Zn alloys promote IEC-6 cells apoptosis, although further experiments will be necessary to formally prove our conclusions. Therefore, the adjustment of the degradation rate is needed for using Mg–Zn alloy as a surgical suture material.

  2. Inhibition of p38 mitogen-activated protein kinase may decrease intestinal epithelial cell apoptosis and improve intestinal epithelial barrier function after ischemia- reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Shu-Yun Zheng; Xiao-Bing Fu; Jian-Guo Xu; Jing-Yu Zhao; Tong-Zhu Sun; Wei Chen

    2005-01-01

    AIM: To investigate the role of p38 mitogen-activated protein kinase in rat small intestine after ischemia-reperfusion (I/R)insult and the relationship between activation of p38 MAPK and apoptotic cell death of intestine.METHODS: Ninety Wistar rats were divided randomly into three groups, namely sham-operated group (C), I/R vehicle group (R) and SB203580 pre-treated group(S).In groups R and S, the superior mesenteric artery(SMA)was separated and occluded for 45 min, then released for reperfusion for0.25, 0.5, 1, 2, 6, 12 and 24 h. In group C, SMA was separated without occlusion. Plasma D-lactate levels were examined and histological changes were observed under a light microscope. The activity of p38 MAPK was determined by Western immunoblotting and apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL).RESULTS: Intestinal ischemia followed by reperfusion activated p38 MAPK, and the maximal level of activation (7.3-fold vs sham-operated group) was reached 30 min after I/R. Treatment with SB 203580, a p38 MAPK inhibitor,reduced intestinal apoptosis (26.72±3.39% vs62.50±3.08%in I/R vehicle, P<0.01) and decreased plasma D-lactate level (0.78±0.15 mmol/L in I/R vehicle vs0.42±0.17 mmol/L in SB-treated group) and improved post-ischemic intestinal histological damage.CONCLUSION: p38 MAPK plays a crucial role in the signal transduction pathway mediating post-ischemic intestinal apoptosis, and inhibition of p38 MAPK may attenuate ischemia-reperfusion injury.

  3. Changes in intestinal glucocorticoid sensitivity in early life shape the risk of epithelial barrier defect in maternal-deprived rats.

    Directory of Open Access Journals (Sweden)

    Nabila Moussaoui

    Full Text Available Glucocorticoids (GC contribute to human intestine ontogeny and accelerate gut barrier development in preparation to birth. Rat gut is immature at birth, and high intestinal GC sensitivity during the first two weeks of life resembles that of premature infants. This makes suckling rats a model to investigate postpartum impact of maternal separation (MS-associated GC release in preterm babies, and whether GC sensitivity may shape MS effects in immature gut. A 4 hours-MS applied once at postnatal day (PND10 enhanced plasma corticosterone in male and female pups, increased by two times the total in vivo intestinal permeability (IP to oral FITC-Dextran 4 kDa (FD4 immediately after the end of MS, and induced bacterial translocation (BT to liver and spleen. Ussing chamber experiments demonstrated a 2-fold increase of permeability to FD4 in the colon immediately after the end of MS, but not in the ileum. Colonic permeability was not only increased for FD4 but also to intact horseradish peroxidase 44 kDa in MS pups. In vivo, the glucocorticoid receptor (GR antagonist RU486 or ML7 blockade of myosin light chain kinase controlling epithelial cytoskeleton contraction prevented MS-induced IP increase to oral FD4 and BT. In addition, the GR agonist dexamethasone dose-dependently mimicked MS-increase of IP to oral FD4. In contrast, MS effects on IP to oral FD4 and BT were absent at PND20, a model for full-term infant, characterized by a marked drop of IP to FD4 in response to dexamethasone, and decreased GR expression in the colon only compared to PND10 pups. These results show that high intestinal GC responsiveness in a rat model of prematurity defines a vulnerable window for a post-delivery MS, evoking immediate disruption of epithelial integrity in the large intestine, and increasing susceptibility to macromolecule passage and bacteremia.

  4. Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death

    International Nuclear Information System (INIS)

    One of the important mechanisms for gastrointestinal (GI) injury following high-dose radiation exposure is apoptosis of epithelial cells. X-linked inhibitor of apoptosis (XIAP) and cellular IAP2 (cIAP2) are intrinsic cellular inhibitors of apoptosis. In order to study the effects of exogenously added IAPs on apoptosis in intestinal epithelial cells, we constructed bacterial expression plasmids containing genes of XIAP (full-length, BIR2 domain and BIR3-RING domain with and without mutations of auto-ubiquitylation sites) and cIAP2 proteins fused to a protein-transduction domain (PTD) derived from HIV-1 Tat protein (TAT) and purified these cell-permeable recombinant proteins. When the TAT-conjugated IAPs were added to rat intestinal epithelial cells IEC6, these proteins were effectively delivered into the cells and inhibited apoptosis, even when added after irradiation. Our results suggest that PTD-mediated delivery of IAPs may have clinical potential, not only for radioprotection but also for rescuing the GI system from radiation injuries. (author)

  5. Cytotoxic Effect and Permeability Activities of Curcumin Analogue; 2, 6-Bis (2, 5-dimethoxybenzy-lidene cyclohexanone (BDMC33 in Caco-2 Cell Model

    Directory of Open Access Journals (Sweden)

    N. Yakubu

    2015-11-01

    Full Text Available Previously, curcumin analogue, 2, 6-bis (2, 5-dimethoxybenzylidene cyclohexanone (BDMC33 with high anti-inflammatory activity was chemically synthesized in our laboratory to enhance the biological activity of curcumin. In this study, the toxicity and permeability activities of 2,6-bis(2,5-dimethoxybenzy-lidenecyclohexanone (BDMC33 in Caco-2 cells was investigated. Toxicity effects using MTT assay and apparent permeability coefficient (Papp, uptake (UR and efflux (ER ratios, and mass balance of BDMC33 after permeation in Caco-2 cells for 180 min were evaluated in apical (A to basolateral (B and basolateral (B to apical (A directions. The similar analyses on 3-(2-fluoro-benzylidene-5-(2-fluorocyclohexylmethylene-piperidin-4-one; (EF-24 (check control were also conducted. The 24 hr LC50 value for BDMC33 and EF-24 on Caco-2 cells were both 50 µM. The Papp value in A→B direction was 3.37 ± 0.47 cm/s (BDMC33 and 2.47 ± 0.15 cm/s (EF-24. Whereas in B→A direction, it was 1.9 ± 0.36 cm/s (BDMC33 and 1.8 ± 0.15 cm/s (EF-24 upon 120 min incubation. The UR and ER ratios calculated were 1.77% and 0.56%, respectively, and the mass balance calculated were 41-44% (BDMC33 and 31-34% (EF-24 in A→B and B→A direction. This study has suggested BDMC33 to be more absorbable than EF-24 in Caco-2 cells. Therefore, BDMC33 could be a leading feature, the anti-inflammatory agent, as it biological activities would be expected outside the intestine.

  6. Multiparametric temporal analysis of the Caco-2/TC7 demonstrated functional and differentiated monolayers as early as 14 days of culture.

    Science.gov (United States)

    Zeller, Perrine; Bricks, Thibault; Vidal, Guillaume; Jacques, Sébastien; Anton, Pauline M; Leclerc, Eric

    2015-05-25

    Reducing the differentiation period for obtaining an in vitro intestinal barrier model is required to reduce the duration and cost for drug screening assays. In this frame, the Caco-2/TC7 subclone differentiation state was investigated from day 0 (D0) to day 32 (D32). As such, the expression of 45 genes (including cell junction, cell polarization, cell functionality, drug transport and metabolism genes) was followed throughout the 32 days. In parallel, the monolayer polarization and the formation of the cellular junctions were characterized by the immuno-staining of occludin, claudin-1 and actin proteins. The cell monolayer permeability was analyzed via transepithelial electric resistance measurements and paracellular transport of Lucifer Yellow. The P-gp efflux efficiency was assessed by rhodamine 123 transport. Alkaline phosphate activity was quantified to assess the cell differentiation. Three stages of differentiation were observed using the clustering of principal component analysis of the RTqPCR data and the overall assays. From D0 to D10, cells were in a proliferation stage and under-differentiated; from D14 to D21 a stable differentiation stage was reached; from D25 to D32 the epithelium seemed to enter into a post-differentiated stage. This study demonstrates that Caco-2/TC7 cells are functional and ready for use in drug screening permeability assays from 14 days in culture when compared with conventional 21 days for Caco-2 cells. In addition, this study provides a refined set of data allowing temporal and multi scale investigations, due to the intracellular kinetics and mRNA levels that can be correlated with membrane protein kinetics and functional extracellular activities. Therefore, shorter time in culture combined with a better knowledge of the cells during the time in culture will in turn help to improve the quality and cost of Caco-2/TC7 assays for drug development. PMID:25725134

  7. Effects of Ursolic Acid Derivatives on Caco-2 Cells and Their Alleviating Role in Streptozocin-Induced Type 2 Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Panpan Wu

    2014-08-01

    Full Text Available In this study, the effect and mechanism of a series of ursolic acid (UA derivatives on glucose uptake were investigated in a Caco-2 cells model. Their effect on hyperglycemia, hyperlipidemia and oxidative stress were also demonstrated in streptozocin (STZ-induced diabetic rats. 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-ylamino]-2-deoxy-glucose (2-NBDG was used as a fluorescein in Caco-2 cells model to screen UA derivatives by glucose uptake and expression of glucose transporter protein (SGLT-1, GLUT-2. Moreover, STZ-induced diabetic rats were administered with these derivatives for 4 weeks of treatment. The fasting blood glucose (FBG, insulin levels, biochemical parameters, lipid levels, and oxidative stress markers were finally evaluated. The results of this study indicated that compounds 10 and 11 significantly inhibited 2-NBDG uptake under both Na+-dependent and Na+-independent conditions by decreasing SGLT-1 and GLUT-2 expression in the Caco-2 cells model. Further in vivo studies revealed that compound 10 significantly reduced hyperglycemia by increasing levels of serum insulin, total protein, and albumin, while the fasting blood glucose, body weight and food intake were restored much closer to those of normal rats. Compounds 10 and 11 showed hypolipidemic activity by decreasing the total amounts of cholesterol (TC and triglycerides (TG. Furthermore, compound 10 showed antioxidant potential which was confirmed by elevation of glutathione (GSH and superoxide dismutase (SOD and reduction of malondialdehyde (MDA levels in the liver and kidney of diabetic rats. It was concluded that compound 10 caused an apparent inhibition of intestinal glucose uptake in Caco-2 cells and hypoglycemia, hypolipidemia and augmented oxidative stress in STZ-induced diabetic rats. Thus, compound 10 could be developed as a potentially complementary therapeutic or prophylactic agent for diabetics mellitus and its complications.

  8. Determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and BALB/c mouse macrophages.

    Science.gov (United States)

    Tamari, Farshad; Tychowski, Joanna; Lorentzen, Laura

    2013-01-01

    The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections. PMID:23748896

  9. The insulin receptor substrate 1 (IRS1 in intestinal epithelial differentiation and in colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Diana L Esposito

    Full Text Available Colorectal cancer (CRC is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1 is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01 and colonic epithelium (P<0.01. Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively. Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin. In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1 shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.

  10. The insulin receptor substrate 1 (IRS1) in intestinal epithelial differentiation and in colorectal cancer.

    Science.gov (United States)

    Esposito, Diana L; Aru, Federica; Lattanzio, Rossano; Morgano, Annalisa; Abbondanza, Michela; Malekzadeh, Reza; Bishehsari, Faraz; Valanzano, Rosa; Russo, Antonio; Piantelli, Mauro; Moschetta, Antonio; Lotti, Lavinia Vittoria; Mariani-Costantini, Renato

    2012-01-01

    Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization. PMID:22558377

  11. Cocoa polyphenols prevent inflammation in the colon of azoxymethane-treated rats and in TNF-α-stimulated Caco-2 cells.

    Science.gov (United States)

    Rodríguez-Ramiro, Ildefonso; Ramos, Sonia; López-Oliva, Elvira; Agis-Torres, Angel; Bravo, Laura; Goya, Luis; Martín, Maria Angeles

    2013-07-28

    Numerous lines of evidence support a relationship between intestinal inflammation and cancer. Therefore, much attention has recently been focused on the identification of natural compounds with anti-inflammatory activities as a strategy to suppress the early stages of colorectal cancer. Because cocoa is a rich source of bioactive compounds, the present study investigated its anti-inflammatory properties in a rat model of azoxymethane (AOM)-induced colon carcinogenesis and in TNF-α-stimulated Caco-2 cells. A total of forty male rats were fed with control or cocoa-enriched diets (12 %) during 8 weeks and injected with saline or AOM (20 mg/kg body weight) during the third and fourth week (n 10 rats/group). At the end of the experiment, colon samples were evaluated for markers of inflammation. The anti-inflammatory activity of a cocoa polyphenolic extract (10 μg/ml) was examined in TNF-α-stimulated Caco-2 cells, an in vitro model of experimentally induced intestinal inflammation. The signalling pathways involved, including NF-κB and the mitogen-activated protein kinase family such as c-Jun NH₂-terminal kinases (JNK), extracellular signal-regulated kinases and p38, were also evaluated. The results show that the cocoa-rich diet decreases the nuclear levels of NF-κB and the expression of pro-inflammatory enzymes such as cyclo-oxygenase-2 and inducible NO synthase induced by AOM in the colon. Additionally, the experiments in Caco-2 cells confirm that cocoa polyphenols effectively down-regulate the levels of inflammatory markers induced by TNF-α by inhibiting NF-κB translocation and JNK phosphorylation. We conclude that cocoa polyphenols suppress inflammation-related colon carcinogenesis and could be promising in the dietary prevention of intestinal inflammation and related cancer development. PMID:23186731

  12. Competitive inhibition of adherence of enterotoxigenic Escherichia coli,enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027

    Institute of Scientific and Technical Information of China (English)

    Shi-Shun Zhong; Zhen-Shu Zhang; Ji-De Wang; Zhuo-Sheng Lai; Qun-Ying Wang; Ling-Jia Pan; Yue-Xin Ren

    2004-01-01

    AIM: To observe competitive inhibition of adherence of enterotoxigenic Escherichia coli(ETEC), enteropathogenic Escherichia coli(EPEC) and Clostridium difficile ( C. difficile)to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027 (B. ado 1027).METHODS: The binding of bacteria to intestinal epithelial cell line Lovo was counted by adhesion assay. The inhibition of adherence of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo by purified adhesin of B. ado 1027was evaluated quantitatively by flow cytometry.RESULTS: The purified adhesin at the concentration of 10μg/mL, 20μg/mL and 30μg/mL except at 1μg/mL and 5μg/mL could inhibit significantly the adhesion of ETEC,EPEC and C. difficile to intestinal epithelial cell line Lovo.Moreover, we observed that a reduction in bacterial adhesion was occurred with increase in the concentration of adhesin,and MFI (Mean fluorescent intensity) was decreased with increase in the concentration of adhesin.CONCLUSION: The purified adhesin of B. ado 1027 can inhibit the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo in a dose-dependent manner.

  13. Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells

    Science.gov (United States)

    Brancaccio, Mariarita; Coretti, Lorena; Florio, Ermanno; Pezone, Antonio; Calabrò, Viola; Falco, Geppino; Keller, Simona; Lembo, Francesca; Avvedimento, Vittorio Enrico; Chiariotti, Lorenzo

    2016-01-01

    Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene. PMID:27253528

  14. Combined assessment of dissolution and epithelial permeability of solid oral dosage forms

    OpenAIRE

    Motz, Stephan

    2007-01-01

    Assessing dissolution and Caco-2 permeability to estimate the in vivo performance of solid oral dosage forms is done since decades. However, permeability assays applying Caco-2 monolayers exhibit the drawback that, in contrast to dissolution testing, a complete dosage form is not eligible for conventional epithelial permeability assays. Hence, an apparatus was developed providing the possibility to assess permeability of solid oral dosage forms based on combination of a Caco-2 monolayer and a...

  15. Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

    DEFF Research Database (Denmark)

    Rosenstiel, Philip; Sina, Christian; End, Caroline;

    2007-01-01

    -kappaB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology......Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes...... intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF...

  16. Analysis of the human intestinal epithelial cell transcriptional response to Lactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactis and Escherichia coli

    DEFF Research Database (Denmark)

    Putaala, H; Barrangou, R; Leyer, G J;

    2010-01-01

    comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33...... regulation of apoptosis and adipogenesis, and lipid-metabolism related regulation by the probiotics. Specific changes such as regulation of cell-cell adhesion by B. lactis 420, superoxide metabolism by L. salivarius Ls-33, and regulation of MAPK pathway by L. acidophilus NCFM™ were noted. Furthermore...

  17. Epithelial barrier and ion transport in coeliac sprue: electrical measurements on intestinal aspiration biopsy specimens.

    OpenAIRE

    Schulzke, J D; Schulzke, I; Fromm, M.; Riecken, E O

    1995-01-01

    Epithelial barrier function and ion transport was studied in coeliac sprue using a miniaturised Ussing device for measurements on diagnostic aspiration biopsy specimens from the jejunum of untreated or gluten free nourished sprue patients, or from healthy controls. Pure epithelial resistance (Re) indicating epithelial barrier function was determined by transmural alternating current impedance analysis. It was reduced by 56% in acute sprue mean (SEM) (9 (1) omega.cm2) compared with controls (2...

  18. Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2.

    Science.gov (United States)

    Dihal, Ashwin A; Woutersen, Ruud A; van Ommen, Ben; Rietjens, Ivonne M C M; Stierum, Rob H

    2006-07-18

    The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001Caco-2 cells formed 5 phase II metabolites, of which the amount of 4'-O-methyl-quercetin-3'-O-glucuronide correlated with the differentiation grade (r=0.99, P<0.003). The increment of cell proliferation at low quercetin concentrations and the decrease in cell differentiation are effects opposite to what would be expected for a functional food ingredient with anti-carcinogenic potential. PMID:16129554

  19. Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells

    OpenAIRE

    Saksena, Seema; Priyamvada, Shubha; Kumar, Anoop; Akhtar, Maria; Soni, Vikas; Anbazhagan, Arivarasu Natarajan; Alakkam, Anas; Alrefai, Waddah A.; Dudeja, Pradeep K.; Gill, Ravinder K.

    2013-01-01

    Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether K...

  20. Folate deprivation induces BCRP (ABCG2) expression and mitoxantrone resistance in Caco-2 cells.

    Science.gov (United States)

    Lemos, Clara; Kathmann, Ietje; Giovannetti, Elisa; Dekker, Henk; Scheffer, George L; Calhau, Conceição; Jansen, Gerrit; Peters, Godefridus J

    2008-10-01

    Folates can induce the expression and activity of the breast-cancer-resistance-protein (BCRP) and the multidrug-resistance-protein-1 (MRP1). Our aim was to study the time-dependent effect of folate deprivation/supplementation on (i) BCRP and MRP expression and (ii) on drug resistance mediated by these transporters. Therefore Caco-2 colon cancer cells usually grown in standard RPMI-medium containing supraphysiological folic acid (FA) concentrations (2.3 muM; high-folate, HF) were gradually adapted to more physiological folate concentrations (1 nM leucovorin (LV) or 1 nM FA; low-folate, LF), resulting in the sublines Caco-2-LF/LV and Caco-2-LF/FA. Caco-2-LF/LV and LF/FA cells exhibited a maximal increase of 5.2- and 9.6-fold for BCRP-mRNA and 3.9- and 5.7-fold for BCRP protein expression, respectively, but no major changes on MRP expression. Overexpression of BCRP in the LF-cells resulted in 3.6- to 6.3-fold resistance to mitoxantrone (MR), which was completely reverted by the BCRP inhibitor Ko143. On the other hand, LF-adapted cells were markedly more sensitive to methotrexate than the HF-counterpart, both after 4-hr (9,870- and 23,923-fold for Caco-2-LF/LV and LF/FA, respectively) and 72-hr (11- and 22-fold for Caco-2-LF/LV and LF/FA, respectively) exposure. Immunofluorescent staining observed with a confocal-laser-scan-microscope revealed that in Caco-2 cells (both HF and LF), BCRP is mainly located in the cytoplasm. In conclusion, folate deprivation induces BCRP expression associated with MR resistance in Caco-2 cells. The intracellular localization of BCRP in these cells suggests that this transporter is not primarily extruding its substrates out of the cell, but rather to an intracellular compartment where folates can be kept as storage. PMID:18623116

  1. Distinctive toxicity of TiO2 rutile/anatase mixed phase nanoparticles on Caco-2 cells.

    Science.gov (United States)

    Gerloff, Kirsten; Fenoglio, Ivana; Carella, Emanuele; Kolling, Julia; Albrecht, Catrin; Boots, Agnes W; Förster, Irmgard; Schins, Roel P F

    2012-03-19

    Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO(2) particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO(2)-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO(2)-toxicity toward intestinal cells. PMID:22263745

  2. Farnesoid X receptor signal is involved in deoxycholic acid-induced intestinal metaplasia of normal human gastric epithelial cells.

    Science.gov (United States)

    Li, Shu; Chen, Xin; Zhou, Lu; Wang, Bang-Mao

    2015-11-01

    The farnesoid X receptor (FXR) signaling pathway is known to be involved in the metabolism of bile acid, glucose and lipid. In the present study, we demonstrated that 400 µmol/l deoxycholic acid (DCA) stimulation promotes the proliferation of normal human gastric epithelial cells (GES-1). In addition, DCA activated FXR and increased the expression of intestinal metaplasia genes, including caudal-related homeobox transcription factor 2 (Cdx2) and mucin 2 (MUC2). The treatment of FXR agonist GW4064/antagonist guggulsterone (Gug.) significantly increased/decreased the expression levels of FXR, Cdx2 and MUC2 protein in DCA-induced GES-1 cells. GW4064/Gug. also enhanced/reduced the nuclear factor-κB (NF-κB) activity and binding of the Cdx2 promoter region and NF-κB, the most common subunit p50 protein. Taken together, the results indicated that DCA is capable of modulating the expression of Cdx2 and the downstream MUC2 via the nuclear receptor FXR-NF-κB activity in normal gastric epithelial cells. FXR signaling pathway may therefore be involved in the intestinal metaplasia of human gastric mucosa. PMID:26324224

  3. Interleukin-17 is a potent immuno-modulator and regulator of normal human intestinal epithelial cell growth

    International Nuclear Information System (INIS)

    Upregulation of the T-cell derived cytokine interleukin (IL-17) was reported in the inflamed intestinal mucosa of patients with inflammatory bowel disorders. In this study, we analyzed the effect of IL-17 on human intestinal epithelial cell (HIEC) turnover and functions. Proliferation and apoptosis in response to IL-17 was monitored in HIEC (cell counts, [3H]thymidine incorporation method, and annexinV-PI-apoptosis assay). Signalling pathways were analyzed by Western blots, electromobility shift assay, and immunofluorescence studies. IL-17 proved to be a potent inhibitor of HIEC proliferation without any pro-apoptotic/necrotic effect. The growth inhibitory effect of IL-17 was mediated via the p38 stress kinase. Consequently, the p38-SAPkinase-inhibitor SB203580 abrogated this anti-mitotic effect. In parallel, IL-17 provoked the degradation of IκBα, allowing nuclear translocation of the p65 NF-κB subunit and induction of the NF-κB-controlled genes IL-6 and -8. IL-17 potently blocks epithelial cell turnover while at the same time amplifying an inflammatory response in a positive feedback manner

  4. New Ways of Thinking about (and Teaching about) Intestinal Epithelial Function

    Science.gov (United States)

    Barrett, Kim E.

    2008-01-01

    This article summarizes a presentation made at the Teaching Refresher Course of the American Physiological Society, which was held at the Experimental Biology meeting in 2007. The intestinal epithelium has important ion transport and barrier functions that contribute pivotally to normal physiological functioning of the intestine and other body…

  5. Chimeric-transgenic mice represent a powerful tool for studying how the proliferation and differentiation programs of intestinal epithelial cell lineages are regulated.

    OpenAIRE

    Hermiston, M L; Green, R. P.; Gordon, J I

    1993-01-01

    An in vivo system has been developed for examining the effects of wild-type or mutant proteins on cell fate determination in the mouse intestinal epithelium or on the proliferation and differentiation programs of its component epithelial lineages. This system takes advantage of the fact that at the conclusion of gut morphogenesis, each intestinal crypt is composed of a monoclonal population of cells descended from a single active multipotent stem cell, each villus is supplied by several monoc...

  6. Anthocyanin absorption and metabolism by human intestinal Caco-2 cells: a review

    OpenAIRE

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

    2015-01-01

    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...

  7. Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

    Directory of Open Access Journals (Sweden)

    Stephanie Plog

    Full Text Available The human CLCA4 (chloride channel regulator, calcium-activated modulates the intestinal phenotype of cystic fibrosis (CF patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

  8. AT1R blocker losartan attenuates intestinal epithelial cell apoptosis in a mouse model of Crohn's disease.

    Science.gov (United States)

    Liu, Tian-Jing; Shi, Yong-Yan; Wang, En-Bo; Zhu, Tong; Zhao, Qun

    2016-02-01

    Angiotensin II, which is the main effector of the renin‑angiotensin system, has an important role in intestinal inflammation via the angiotensin II type 1 receptor (AT1R). The present study aimed to investigate the protective effects of the AT1R blocker losartan on 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis. Losartan was administered to male adult C57BL/6 J mice 2 weeks prior to the induction of colitis, and images of the whole colon were captured to record changes, scored according to a microscopic scoring system, and reverse transcription-quantitative polymerase chain reaction were performed in order to investigate colonic inflammation. In addition, intestinal epithelial barrier permeability was evaluated, and intestinal epithelial cell (IEC) apoptosis was measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and apoptosis-related protein expression levels were detected by western blotting. Losartan was able to attenuate TNBS-induced body weight loss and colonic damage. Furthermore, T helper 1-mediated proinflammatory cytokines were suppressed by losartan, and gut permeability was largely preserved. TUNEL staining revealed reduced IEC apoptosis in the losartan-treated mice. Losartan also increased the B-cell lymphoma 2 (Bcl2)/Bcl-2-associated X protein (Bax) ratio and suppressed caspase-3 induction. These results suggested that the AT1R blocker losartan may attenuate TNBS-induced colitis by inhibiting the apoptosis of IECs. The effects of losartan were partially mediated through increasing the Bcl-2/Bax ratio and subsequently suppressing the induction of the proapoptotic mediator caspase-3. PMID:26676112

  9. Ephrin-B reverse signaling induces expression of wound healing associated genes in IEC-6 intestinal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Christian Hafner; Stefanie Meyer; Ilja Hagen; Bernd Becker; Alexander Roesch; Michael Landthaler; Thomas Vogt

    2005-01-01

    AIM: Eph receptors and ephrin ligands play a pivotal role in development and tissue maintenance. Since previous data have indicated an involvement of ephrin-B2 in epithelial healing, we investigated the gene expression and downstream signaling pathways induced by ephrin-B mediated cell-cell signaling in intestinal epithelial cells.METHODS: Upon stimulation of ephrin-B pathways in IFC-6 cells with recombinant rat EphB1-Fc, gene expression was analyzed by Affymetrix(R) rat genome 230 high density arrays at different time points. Differentially expressed genes were confirmed by real-time RT-PCR. In addition, MAP kinase pathways and focal adhesion kinase (FAK) activation downstream of ephrin-B were investigated by immunoblotting and fluorescence microscopy.RESULTS: Stimulation of the ephrin-B reverse signaling pathway in IEC-6 cells induces predominant expression of genes known to be involved into wound healing/cell migration, antiapoptotic pathways, host defense and inflammation. Cox-2, c-Fos, Egr-1, Egr-2, and MCP-1 were found among the most significantly regulated genes.Furthermore, we show that the expression of repairrelated genes is also accompanied by activation of the ERK1/2 MAP kinase pathway and FAK, two key regulators of epithelial restitution.CONCLUSION: Stimulation of the ephrin-B reverse signaling pathway induces a phenotype characterized by upregulation of repair-related genes, which may partially be mediated by ERK1/2 pathways.

  10. Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells.

    Science.gov (United States)

    Estevez, Ana Y; Roberts, Randolph K; Strange, Kevin

    2003-08-01

    The nematode Caenorhabditis elegans offers significant experimental advantages for defining the genetic basis of diverse biological processes. Genetic and physiological analyses have demonstrated that inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in intestinal epithelial cells play a central role in regulating the nematode defecation cycle, an ultradian rhythm with a periodicity of 45-50 s. Patch clamp studies combined with behavioral assays and forward and reverse genetic screening would provide a powerful approach for defining the molecular details of oscillatory Ca2+ signaling. However, electrophysiological characterization of the intestinal epithelium has not been possible because of its relative inaccessibility. We developed primary intestinal epithelial cell cultures that circumvent this problem. Intestinal cells express two highly Ca2+-selective, voltage-independent conductances. One conductance, IORCa, is constitutively active, exhibits strong outward rectification, is 60-70-fold more selective for Ca2+ than Na+, is inhibited by intracellular Mg2+ with a K1/2 of 692 microM, and is insensitive to Ca2+ store depletion. Inhibition of IORCa with high intracellular Mg2+ concentrations revealed the presence of a small amplitude conductance that was activated by passive depletion of intracellular Ca2+ stores. Active depletion of Ca2+ stores with IP3 or ionomycin increased the rate of current activation approximately 8- and approximately 22-fold compared with passive store depletion. The store-operated conductance, ISOC, exhibits strong inward rectification, and the channel is highly selective for Ca2+ over monovalent cations with a divalent cation selectivity sequence of Ca2+ > Ba2+ approximately Sr2+. Reversal potentials for ISOC could not be detected accurately between 0 and +80 mV, suggesting that PCa/PNa of the channel may exceed 1,000:1. Lanthanum, SKF 96365, and 2-APB inhibit both IORCa and ISOC reversibly. Our studies provide the first

  11. Low molecular weight heparin nanoparticles: mucoadhesion and behaviour in Caco-2 cells

    Science.gov (United States)

    Lamprecht, Alf; Koenig, Petra; Ubrich, Nathalie; Maincent, Philippe; Neumann, Dirk

    2006-08-01

    Nanoparticles (NPs) have shown their efficiency in increasing the oral bioavailability of macromolecular drugs, among them heparin. However, mechanisms of absorption are still unclear. Here, heparin-loaded NPs were prepared from different polymers (Eudragit® RS, poly(lactic-co-glycolic acid) (PLGA), and their respective mixtures) and analysed for their mucoadhesive properties using a resonant mirror system. Subsequent binding and drug transport studies of the free heparin and heparin-loaded NPs were carried out on Caco-2 cells. Cationic NPs were found to be mucoadhesive, while pure drug and polyester NPs were not. The adsorption of anionic heparin masked the positive surface charge of the particles, thus partially diminishing the adhesiveness to mucin. Increased binding to Caco-2 cells was found for all NP formulation, with RS/PLGA NPs showing maximum binding. However, the transport of heparin was the same for the RS/PLGA NPs and the PLGA NPs and slightly higher than for the free drug. In all cases, no NP transport across the cell layer was observed. When Caco-2 cells were coated with an additional mucin layer, cell binding of RS NPs and RS/PLGA NPs was further increased. Transport across Caco-2 cells demonstrated similar tendencies to results obtained without mucin. In contrast, cationic NPs led to higher heparin transport in the presence of mucin. The mechanism of drug absorption associated with RS NPs was concluded to be independent of typical transcellular NP transport.

  12. Low molecular weight heparin nanoparticles: mucoadhesion and behaviour in Caco-2 cells

    International Nuclear Information System (INIS)

    Nanoparticles (NPs) have shown their efficiency in increasing the oral bioavailability of macromolecular drugs, among them heparin. However, mechanisms of absorption are still unclear. Here, heparin-loaded NPs were prepared from different polymers (Eudragit[reg] RS, poly(lactic-co-glycolic acid) (PLGA), and their respective mixtures) and analysed for their mucoadhesive properties using a resonant mirror system. Subsequent binding and drug transport studies of the free heparin and heparin-loaded NPs were carried out on Caco-2 cells. Cationic NPs were found to be mucoadhesive, while pure drug and polyester NPs were not. The adsorption of anionic heparin masked the positive surface charge of the particles, thus partially diminishing the adhesiveness to mucin. Increased binding to Caco-2 cells was found for all NP formulation, with RS/PLGA NPs showing maximum binding. However, the transport of heparin was the same for the RS/PLGA NPs and the PLGA NPs and slightly higher than for the free drug. In all cases, no NP transport across the cell layer was observed. When Caco-2 cells were coated with an additional mucin layer, cell binding of RS NPs and RS/PLGA NPs was further increased. Transport across Caco-2 cells demonstrated similar tendencies to results obtained without mucin. In contrast, cationic NPs led to higher heparin transport in the presence of mucin. The mechanism of drug absorption associated with RS NPs was concluded to be independent of typical transcellular NP transport

  13. Low molecular weight heparin nanoparticles: mucoadhesion and behaviour in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lamprecht, Alf [Laboratory of Pharmaceutical Engineering, University of Besancon (France); Koenig, Petra [Center for Bioinformatics, Saarland University, Saarbruecken (Germany); Ubrich, Nathalie [Laboratory of Pharmaceutical Technology, University of Nancy (France); Maincent, Philippe [Laboratory of Pharmaceutical Technology, University of Nancy (France); Neumann, Dirk [Center for Bioinformatics, Saarland University, Saarbruecken (Germany)

    2006-08-14

    Nanoparticles (NPs) have shown their efficiency in increasing the oral bioavailability of macromolecular drugs, among them heparin. However, mechanisms of absorption are still unclear. Here, heparin-loaded NPs were prepared from different polymers (Eudragit[reg] RS, poly(lactic-co-glycolic acid) (PLGA), and their respective mixtures) and analysed for their mucoadhesive properties using a resonant mirror system. Subsequent binding and drug transport studies of the free heparin and heparin-loaded NPs were carried out on Caco-2 cells. Cationic NPs were found to be mucoadhesive, while pure drug and polyester NPs were not. The adsorption of anionic heparin masked the positive surface charge of the particles, thus partially diminishing the adhesiveness to mucin. Increased binding to Caco-2 cells was found for all NP formulation, with RS/PLGA NPs showing maximum binding. However, the transport of heparin was the same for the RS/PLGA NPs and the PLGA NPs and slightly higher than for the free drug. In all cases, no NP transport across the cell layer was observed. When Caco-2 cells were coated with an additional mucin layer, cell binding of RS NPs and RS/PLGA NPs was further increased. Transport across Caco-2 cells demonstrated similar tendencies to results obtained without mucin. In contrast, cationic NPs led to higher heparin transport in the presence of mucin. The mechanism of drug absorption associated with RS NPs was concluded to be independent of typical transcellular NP transport.

  14. Caco-2 cell monolayer integrity and effect of probiotic Escherichia coli Nissle 1917 components

    Czech Academy of Sciences Publication Activity Database

    Štětinová, V.; Smetanová, L.; Květina, J.; Svoboda, Z.; Zídek, Zdeněk; Tlaskalová-Hogenová, Helena

    2010-01-01

    Roč. 31, - (2010), s. 51-56. ISSN 0172-780X R&D Projects: GA ČR GA305/08/0535; GA MZd NS9775 Institutional support: RVO:68378041 ; RVO:61388971 Keywords : probiotic s * lipopolysaccharide (LPS) * Caco-2 cells Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 1.621, year: 2010

  15. Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease.

    Science.gov (United States)

    Mathewson, Nathan D; Jenq, Robert; Mathew, Anna V; Koenigsknecht, Mark; Hanash, Alan; Toubai, Tomomi; Oravecz-Wilson, Katherine; Wu, Shin-Rong; Sun, Yaping; Rossi, Corinne; Fujiwara, Hideaki; Byun, Jaeman; Shono, Yusuke; Lindemans, Caroline; Calafiore, Marco; Schmidt, Thomas C; Honda, Kenya; Young, Vincent B; Pennathur, Subramaniam; van den Brink, Marcel; Reddy, Pavan

    2016-05-01

    The effect of alterations in intestinal microbiota on microbial metabolites and on disease processes such as graft-versus-host disease (GVHD) is not known. Here we carried out an unbiased analysis to identify previously unidentified alterations in gastrointestinal microbiota-derived short-chain fatty acids (SCFAs) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amount of only one SCFA, butyrate, were observed only in the intestinal tissue. The reduced butyrate in CD326(+) intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored after local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate-producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate disease severity. PMID:26998764

  16. Glucose and Palmitate Differentially Regulate PFKFB3/iPFK2 and Inflammatory Responses in Mouse Intestinal Epithelial Cells

    Science.gov (United States)

    Botchlett, Rachel; Li, Honggui; Guo, Xin; Qi, Ting; Zhao, JiaJia; Zheng, Juan; Woo, Shih-Lung; Pei, Ya; Liu, Mengyang; Hu, Xiang; Chen, Guang; Guo, Ting; Yang, Sijun; Li, Qifu; Xiao, Xiaoqiu; Huo, Yuqing; Wu, Chaodong

    2016-01-01

    The gene PFKFB3 encodes for inducible 6-phosphofructo-2-kinase, a glycolysis-regulatory enzyme that protects against diet-induced intestine inflammation. However, it is unclear how nutrient overload regulates PFKFB3 expression and inflammatory responses in intestinal epithelial cells (IECs). In the present study, primary IECs were isolated from small intestine of C57BL/6J mice fed a low-fat diet (LFD) or high-fat diet (HFD) for 12 weeks. Additionally, CMT-93 cells, a cell line for IECs, were cultured in low glucose (LG, 5.5 mmol/L) or high glucose (HG, 27.5 mmol/L) medium and treated with palmitate (50 μmol/L) or bovine serum albumin (BSA) for 24 hr. These cells were analyzed for PFKFB3 and inflammatory markers. Compared with LFD, HFD feeding decreased IEC PFKFB3 expression and increased IEC proinflammatory responses. In CMT-93 cells, HG significantly increased PFKFB3 expression and proinflammatory responses compared with LG. Interestingly, palmitate decreased PFKFB3 expression and increased proinflammatory responses compared with BSA, regardless of glucose concentrations. Furthermore, HG significantly increased PFKFB3 promoter transcription activity compared with LG. Upon PFKFB3 overexpression, proinflammatory responses in CMT-93 cells were decreased. Taken together, these results indicate that in IECs glucose stimulates PFKFB3 expression and palmitate contributes to increased proinflammatory responses. Therefore, PFKFB3 regulates IEC inflammatory status in response to macronutrients. PMID:27387960

  17. Polyphenol-Rich Propolis Extracts Strengthen Intestinal Barrier Function by Activating AMPK and ERK Signaling.

    Science.gov (United States)

    Wang, Kai; Jin, Xiaolu; Chen, Yifan; Song, Zehe; Jiang, Xiasen; Hu, Fuliang; Conlon, Michael A; Topping, David L

    2016-01-01

    Propolis has abundant polyphenolic constituents and is used widely as a health/functional food. Here, we investigated the effects of polyphenol-rich propolis extracts (PPE) on intestinal barrier function in human intestinal epithelial Caco-2 cells, as well as in rats. In Caco-2 cells, PPE increased transepithelial electrical resistance and decreased lucifer yellow flux. PPE-treated cells showed increased expression of the tight junction (TJ) loci occludin and zona occludens (ZO)-1. Confocal microscopy showed organized expressions in proteins related to TJ assembly, i.e., occludin and ZO-1, in response to PPE. Furthermore, PPE led to the activation of AMPK, ERK1/2, p38, and Akt. Using selective inhibitors, we found that the positive effects of PPE on barrier function were abolished in cells in which AMPK and ERK1/2 signaling were inhibited. Moreover, rats fed a diet supplemented with PPE (0.3% in the diet) exhibited increased colonic epithelium ZO-1 expression. Overall, these data suggest that PPE strengthens intestinal barrier function by activating AMPK and ERK signaling and provide novel insights into the potential application of propolis for human gut health. PMID:27164138

  18. Polyphenol-Rich Propolis Extracts Strengthen Intestinal Barrier Function by Activating AMPK and ERK Signaling

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2016-05-01

    Full Text Available Propolis has abundant polyphenolic constituents and is used widely as a health/functional food. Here, we investigated the effects of polyphenol-rich propolis extracts (PPE on intestinal barrier function in human intestinal epithelial Caco-2 cells, as well as in rats. In Caco-2 cells, PPE increased transepithelial electrical resistance and decreased lucifer yellow flux. PPE-treated cells showed increased expression of the tight junction (TJ loci occludin and zona occludens (ZO-1. Confocal microscopy showed organized expressions in proteins related to TJ assembly, i.e., occludin and ZO-1, in response to PPE. Furthermore, PPE led to the activation of AMPK, ERK1/2, p38, and Akt. Using selective inhibitors, we found that the positive effects of PPE on barrier function were abolished in cells in which AMPK and ERK1/2 signaling were inhibited. Moreover, rats fed a diet supplemented with PPE (0.3% in the diet exhibited increased colonic epithelium ZO-1 expression. Overall, these data suggest that PPE strengthens intestinal barrier function by activating AMPK and ERK signaling and provide novel insights into the potential application of propolis for human gut health.

  19. Influence of charge on FITC-BSA-loaded chondroitin sulfate-chitosan nanoparticles upon cell uptake in human Caco-2 cell monolayers

    Directory of Open Access Journals (Sweden)

    Hu CS

    2012-09-01

    Full Text Available Chieh-shen Hu,1 Chiao-hsi Chiang,2 Po-da Hong,1,4,* Ming-kung Yeh1–3,*1Biomedical Engineering Program, Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology; 2School of Pharmacy, National Defence Medical Center; 3Bureau of Pharmaceutical Affairs, Ministry of National Defence Medical Affairs Bureau; 4Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taiwan, Republic of China*These authors contributed equally to this workBackground and methods: Chondroitin sulfate-chitosan (ChS-CS nanoparticles and positively and negatively charged fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA-loaded ChS-CS nanoparticles were prepared and characterized. The properties of ChS-CS nanoparticles, including cellular uptake, cytotoxicity, and transepithelial transport, as well as findings on field emission-scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy were evaluated in human epithelial colorectal adenocarcinoma (Caco-2 fibroblasts. ChS-CS nanoparticles with a mean particle size of 250 nm and zeta potentials ranging from –30 to +18 mV were prepared using an ionic gelation method.Results: Standard cell viability assays demonstrated that cells incubated with ChS-CS and FITC-BSA-loaded ChS-CS nanoparticles remained more than 95% viable at particle concentrations up to 0.1 mg/mL. Endocytosis of nanoparticles was confirmed by confocal laser scanning microscopy and measured by flow cytometry. Ex vivo transepithelial transport studies using Caco-2 cells indicated that the nanoparticles were effectively transported into Caco-2 cells via endocytosis. The uptake of positively charged FITC-BSA-loaded ChS-CS nanoparticles across the epithelial membrane was more efficient than that of the negatively charged nanoparticles.Conclusion: The ChS-CS nanoparticles fabricated in this study were

  20. 3-D intestinal scaffolds for evaluating the therapeutic potential of probiotics.

    Science.gov (United States)

    Costello, Cait M; Sorna, Rachel M; Goh, Yih-Lin; Cengic, Ivana; Jain, Nina K; March, John C

    2014-07-01

    Biomimetic in vitro intestinal models are becoming useful tools for studying host-microbial interactions. In the past, these models have typically been limited to simple cultures on 2-D scaffolds or Transwell inserts, but it is widely understood that epithelial cells cultured in 3-D environments exhibit different phenotypes that are more reflective of native tissue, and that different microbial species will preferentially adhere to select locations along the intestinal villi. We used a synthetic 3-D tissue scaffold with villous features that could support the coculture of epithelial cell types with select bacterial populations. Our end goal was to establish microbial niches along the crypt-villus axis in order to mimic the natural microenvironment of the small intestine, which could potentially provide new insights into microbe-induced intestinal disorders, as well as enabling targeted probiotic therapies. We recreated the surface topography of the small intestine by fabricating a biodegradable and biocompatible villous scaffold using poly lactic-glycolic acid to enable the culture of Caco-2 with differentiation along the crypt-villus axis in a similar manner to native intestines. This was then used as a platform to mimic the adhesion and invasion profiles of both Salmonella and Pseudomonas, and assess the therapeutic potential of Lactobacillus and commensal Escherichia coli in a 3-D setting. We found that, in a 3-D environment, Lactobacillus is more successful at displacing pathogens, whereas Nissle is more effective at inhibiting pathogen adhesion. PMID:24798584

  1. The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression

    International Nuclear Information System (INIS)

    'The gastrointestinal tract represents the first barrier against food contaminants as well as the first target for these toxicants. Deoxynivalenol (DON) is a mycotoxin that commonly contaminates cereals and causes various toxicological effects. Through consumption of contaminated cereals and cereal products, human and pigs are exposed to this mycotoxin. Using in vitro, ex vivo and in vivo approaches, we investigated the effects of DON on the intestinal epithelium. We demonstrated that, in intestinal epithelial cell lines from porcine (IPEC-1) or human (Caco-2) origin, DON decreases trans-epithelial electrical resistance (TEER) and increases in a time and dose-dependent manner the paracellular permeability to 4 kDa dextran and to pathogenic Escherichia coli across intestinal cell monolayers. In pig explants treated with DON, we also observed an increased permeability of intestinal tissue. These alterations of barrier function were associated with a specific reduction in the expression of claudins, which was also seen in vivo in the jejunum of piglets exposed to DON-contaminated feed. In conclusion, DON alters claudin expression and decreases the barrier function of the intestinal epithelium. Considering that high levels of DON may be present in food or feed, consumption of DON-contaminated food/feed may induce intestinal damage and has consequences for human and animal health.

  2. Human intestinal epithelial cells in innate immunity : interactions with normal microbiota and pathogenic bacteria

    OpenAIRE

    Ou, Gangwei

    2009-01-01

    Rod-shaped bacteria were previously shown to be associated with the small intestinal epithelium of children with celiac disease (CD). Using culture-dependent and independent methods, we characterized the microbiota of small intestine in children with CD and controls. The normal microbiota constitutes an unique organ-specific biofilm. Dominant bacteria are Streptococcus, Neisseria, Veillonella, Gemella, Actinomyces, Rothia and Haemophilus. Altogether 162 Genus Level Operational Taxonomic Units...

  3. Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis

    OpenAIRE

    Perrone, Erin E.; Jung, Enjae; Breed, Elise; Dominguez, Jessica A.; Liang, Zhe; Clark, Andrew T.; Dunne, W. Michael; Burd, Eileen M.; Coopersmith, Craig M.

    2012-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and sacrificed 24 hours later. Septic animals had a marked increas...

  4. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization

    International Nuclear Information System (INIS)

    Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

  5. Milk peptides increase iron solubility in water but do not affect DMT-1 expression in Caco-2 cells

    Science.gov (United States)

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. Our objectives were to investigate whether these fractions a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells b) enhance iron dialyzability when added in meals. Peptid...

  6. Anti-inflammatory activity of the basolateral fraction of Caco-2 cells exposed to a rosemary supercritical extract

    NARCIS (Netherlands)

    Arranz, E.; Mes, J.J.; Wichers, H.J.; Jaime, L.; Reglero, G.; Santoyo, S.

    2015-01-01

    The anti-inflammatory activity of the basolateral fraction of Caco-2 cells exposed to a rosemary supercritical extract was examined. Uptake of rosemary extract fractions was tested on Caco-2 cell monolayers (2–12 h incubation times) and the quantification of carnosic acid and carnosol was performed

  7. Physiological Concentration of Exogenous Lactate Reduces Antimycin A Triggered Oxidative Stress in Intestinal Epithelial Cell Line IPEC-1 and IPEC-J2 In Vitro

    Science.gov (United States)

    Kahlert, Stefan; Junnikkala, Sami; Renner, Lydia; Hynönen, Ulla; Hartig, Roland; Nossol, Constanze; Barta-Böszörményi, Anikó; Dänicke, Sven; Souffrant, Wolfgang-Bernhard; Palva, Airi; Rothkötter, Hermann-Josef; Kluess, Jeannette

    2016-01-01

    Weaning triggers an adaptation of the gut function including luminal lactate generation by lactobacilli, depending on gastrointestinal site. We hypothesized that both lactobacilli and lactate influence porcine intestinal epithelial cells. In vivo experiments showed that concentration of lactate was significantly higher in gastric, duodenal and jejunal chyme of suckling piglets compared to their weaned counterparts. In an in vitro study we investigated the impact of physiological lactate concentration as derived from the in vivo study on the porcine intestinal epithelial cells IPEC-1 and IPEC-J2. We detected direct adherence of lactobacilli on the apical epithelial surface and a modulated F-actin structure. Application of lactobacilli culture supernatant alone or lactate (25 mM) at low pH (pH 4) changed the F-actin structure in a similar manner. Treatment of IPEC cultures with lactate at near neutral pH resulted in a significantly reduced superoxide-generation in Antimycin A-challenged cells. This protective effect was nearly completely reversed by inhibition of cellular lactate uptake via monocarboxylate transporter. Lactate treatment enhanced NADH autofluorescence ratio (Fcytosol/Fnucleus) in non-challenged cells, indicating an increased availability of reduced nucleotides, but did not change the overall ATP content of the cells. Lactobacilli-derived physiological lactate concentration in intestine is relevant for alleviation of redox stress in intestinal epithelial cells. PMID:27054581

  8. Influence of the intrinsic gut microbiota on transcriptional regulation of genes involved in the early life development of intestinal epithelial integrity

    DEFF Research Database (Denmark)

    Bergström, Anders; Kristensen, Matilde Bylov; Frøkjær, Hanne;

    2010-01-01

    The interplay between the gut microbiota and the integrity of the intestinal mucus layer is important both in the maintenance of the epithelial barrier as part of the innate immune defense, and in the conservation of gut homeostasis. Interesting parameters are the mucins, which protect the mucosal...

  9. Conjugated primary bile salts reduce permeability of endotoxin through intestinal epithelial cells and synergize with phosphatidylcholine in suppression of inflammatory cytokine production

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Schaeckeler, S.; Moser, L.;

    2007-01-01

    OBJECTIVE: Endotoxemia was shown to be integral in the pathophysiology of obstructive jaundice. In the current study, the role of conjugated primary bile salts (CPBS) and phosphatidylcholine on the permeability of endotoxin through a layer of intestinal epithelial cells and the consequent...

  10. Conjugated primary bile salts reduce permeability of endotoxin through bacteria-stimulated intestinal epithelial cells and synergize with lecithin in suppression of inflammatory cytokine production

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Schaeckeler, Simone; Moser, Lydia;

    2007-01-01

    Objective: Endotoxemia was shown to be integral in the pathophysiology of obstructive jaundice. In the current study, the role of conjugated primary bile salts (CPBS) and phosphatidylcholine on the permeability of endotoxin through a layer of intestinal epithelial cells and the consequent activat...

  11. Effect of CpG-ODN combined with radiation on micronuclei cell of the human intestinal crypt epithelial cell

    International Nuclear Information System (INIS)

    In order study the changes of micronuclei cell frequency in the non-immune cell types, the human intestinal crypt epithelial cell (HIEC) was treated by CpG-ODN after radiation. MTT assay and micronuclei assay were used in this research. The result of MTT assay shows that CpG-ODN does not have any toxicity to HIEC in the concentration range of 0.00-1.25 μmol/L. Micronuclei assay measurement indicates that CpG-ODN can protect HIEC from radiation damage by reducing the micronucleus frequency (MNF) and the micronucleus cell frequency (MNCF). The experiment results reveal that CpG-ODN is safe and may have radioprotection effect on some non-immune human cell types. (authors)

  12. Analysis of the human intestinal epithelial cell transcriptional response to Lactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactis and Escherichia coli.

    Science.gov (United States)

    Putaala, H; Barrangou, R; Leyer, G J; Ouwehand, A C; Hansen, E Bech; Romero, D A; Rautonen, N

    2010-09-01

    The complex microbial population residing in the human gastrointestinal tract consists of commensal, potential pathogenic and beneficial species, which are probably perceived differently by the host and consequently could be expected to trigger specific transcriptional responses. Here, we provide a comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33 DCE-induced changes were overall more similar to those of B. lactis 420 than to L. acidophilus NCFM™, which is consistent with previously observed in vivo immunomodulation properties. In the gene ontology and pathway analyses both specific and unspecific changes were observed. Common to all was the regulation of apoptosis and adipogenesis, and lipid-metabolism related regulation by the probiotics. Specific changes such as regulation of cell-cell adhesion by B. lactis 420, superoxide metabolism by L. salivarius Ls-33, and regulation of MAPK pathway by L. acidophilus NCFM™ were noted. Furthermore, fundamental differences were observed between the pathogenic and probiotic treatments in the Toll-like receptor pathway, especially for adapter molecules with a lowered level of transcriptional activation of MyD88, TRIF, IRAK1 and TRAF6 by probiotics compared to EHEC. The results in this study provide insights into the relationship between probiotics and human intestinal epithelial cells, notably with regard to strain-specific responses, and highlight the differences between transcriptional responses to pathogenic and probiotic bacteria. PMID:21831765

  13. Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Murano, Tatsuro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Okamoto, Ryuichi, E-mail: rokamoto.gast@tmd.ac.jp [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Go; Nakata, Toru; Hibiya, Shuji; Shimizu, Hiromichi; Fujii, Satoru; Kano, Yoshihito; Mizutani, Tomohiro; Yui, Shiro; Akiyama-Morio, Junko; Nemoto, Yasuhiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Tsuchiya, Kiichiro; Nakamura, Tetsuya [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan)

    2014-01-17

    Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

  14. Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

    International Nuclear Information System (INIS)

    Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs

  15. Development of relationship between intestinal commensal bacteria and intestinal epithelial barrier%肠道共生细菌和肠上皮屏障间关系的研究进展

    Institute of Scientific and Technical Information of China (English)

    施君

    2010-01-01

    After birth,human intestinal tract mucosa is exposed to a large community of commensal and pathogenic bacteria. As a first line of defense,the intestinal epithelial barrier (IEB) kills the pathogen by signaling to the innate immune system, through pattern recognition receptors, while it produces protective respond towards the commensal bacteria. Intestinal epithelial cells play an important role in forming immune tolerance to the commensal bacteria and make intestinal homeostasis. Commensal bacteria can resist the pathogenic bacteria invasion. The signals of commensal are required for development of intestinal epithelial barrier and intestinal innate and adaptive immunity. It is essential for the host to have a balance between the commensal bacteria and intestinal tract,once the balance is broken, the intestinal inflammation disease will be caused. Thus ,this review will discuss the relationship between intestinal commensal bacteria and intestinal epithelial barrier in several aspects, such as the role of the commensal bacteria, the mechanism of producing commensal tolerance by IEB and the disease caused by imbalance between the commensal and IEB.%人类出生后,其胃肠道黏膜表面与肠道共生细菌和致病性病原体密切接触.肠道上皮屏障作为抵御细菌入侵的第一道防线,通过模式识别受体产生对致病性病原体杀伤性免疫应答,而对共生细菌产生保护性应答.肠上皮细胞在对共生细菌形成免疫耐受,维持肠道免疫稳态中发挥重要作用.共生细菌能协助肠道上皮抵御病原体侵袭,并调节肠道免疫发育和免疫功能.在共生细菌和宿主肠道之间形成免疫平衡,否则易引起肠道炎症疾病.该文从共生细菌对宿主肠道的作用、肠上皮屏障对共生细菌形成免疫耐受机制以及肠道上皮屏障对共生细菌识别平衡破坏引起的疾病等多方面对共生细菌和肠上皮屏障之间关系作一综述.

  16. Mechanisms of lysophosphatidic acid (LPA) mediated stimulation of intestinal apical Cl-/OH- exchange.

    Science.gov (United States)

    Singla, Amika; Dwivedi, Alka; Saksena, Seema; Gill, Ravinder K; Alrefai, Waddah A; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

    2010-02-01

    Lysophosphatidic acid (LPA), a potent bioactive phospholipid, is a natural component of food products like soy and egg yolk. LPA modulates a number of epithelial functions and has been shown to inhibit cholera toxin-induced diarrhea. Antidiarrheal effects of LPA are known to be mediated by inhibiting chloride secretion. However, the effects of LPA on chloride absorption in the mammalian intestine are not known. The present studies examined the effects of LPA on apical Cl(-)/OH(-) exchangers known to be involved in chloride absorption in intestinal epithelial cells. Caco-2 cells were treated with LPA, and Cl(-)/OH(-) exchange activity was measured as DIDS-sensitive (36)Cl(-) uptake. Cell surface biotinylation studies were performed to evaluate the effect of LPA on cell surface levels of apical Cl(-)/OH(-) exchangers, downregulated in adenoma (DRA) (SLC26A3), and putative anion transporter-1 (SLC26A6). Treatment of Caco-2 cells with LPA (100 muM) significantly stimulated Cl(-)/OH(-) exchange activity. Specific agonist for LPA2 receptor mimicked the effects of LPA. LPA-mediated stimulation of Cl(-)/OH(-) exchange activity was dependent on activation of phosphatidylinositol 3-kinase/Akt signaling pathway. Consistent with the functional activity, LPA treatment resulted in increased levels of DRA on the apical membrane. Our results demonstrate that LPA stimulates apical Cl(-)/OH(-) exchange activity and surface levels of DRA in intestinal epithelial cells. This increase in Cl(-)/OH(-) exchange may contribute to the antidiarrheal effects of LPA. PMID:19910524

  17. miRNAs modified by dietary lipids in Caco-2 cells. A microarray screening

    Directory of Open Access Journals (Sweden)

    Lidia Daimiel

    2015-09-01

    Full Text Available We performed a screening of miRNAs regulated by dietary lipids in a cellular model of enterocytes, Caco-2 cells. Our aim was to describe new lipid-modified miRNAs with an implication in lipid homeostasis and cardiovascular disease [1,2]. For that purpose, we treated differentiated Caco-2 cells with micelles containing the assayed lipids (cholesterol, conjugated linoleic acid and docosahexaenoic acid and the screening of miRNAs was carried out by microarray using the μParaflo®Microfluidic Biochip Technology of LC Sciences (Huston, TX, USA. Experimental design, microarray description and raw data have been made available in the GEO database with the reference number of GSE59153. Here we described in detail the experimental design and methods used to obtain the relative expression data.

  18. Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

    Science.gov (United States)

    Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

    2015-04-01

    Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell-cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens. PMID:25847917

  19. Permeation of roxarsone and its metabolites increases caco-2 cell proliferation

    OpenAIRE

    2013-01-01

    The benzenearsonate, Roxarsone, has been used since 1944 as an antimicrobial, growth-promoting poultry feed additive. USGS and EPA report that Roxarsone (4-hydroxy-3-nitrobenzenearsonate) and metabolites, including AHBA (3-amino-4-hydroxybenzenearsonate), contaminate waterways at greater than 1100 tons annually. To assess human impact of these organic arsenic water contaminants, it was important to study their potential absorption. The human adenocarcinoma cell line, Caco-2, is a model for in...

  20. High glucose concentration in isotonic media alters Caco-2 cell permeability

    OpenAIRE

    Souza, Vanessa M. D; Shertzer, Howard G.; Menon, Anil G.; Pauletti, Giovanni M.

    2003-01-01

    Caco-2 cell permeability was evaluated in isotonic media containing high (25mM) or physiological (5.5mM) glucose concentrations. Transepithelial electrical resistance (TEER) and membrane fluidity were measured to assess glucose-induced alterations in physical barrier properties. In parallel, distribution of the actin filament (F-actin) and zonula occludens-1 (ZO-1) proteins was assessed by confocal microscopy. Transepithelial fluxes of mannitol, hydrocortisone, digoxin, and glycyl sarcosine (...

  1. Metabolites produced by probiotic Lactobacilli rapidly increase glucose uptake by Caco-2 cells

    OpenAIRE

    Buddington Randal K; Kimura Yasuhiro; Rooj Arun K

    2010-01-01

    Abstract Background Although probiotic bacteria and their metabolites alter enterocyte gene expression, rapid, non-genomic responses have not been examined. The present study measured accumulation of tracer (2 μM) glucose by Caco-2 cells after exposure for 10 min or less to a chemically defined medium (CDM) with different monosaccharides before and after anaerobic culture of probiotic Lactobacilli. Results Growth of L. acidophilus was supported by CDM with 110 mM glucose, fructose, and mannos...

  2. A Potential Daidzein Derivative Enhances Cytotoxicity of Epirubicin on Human Colon Adenocarcinoma Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Yu-Li Lo

    2012-12-01

    Full Text Available In this study, we evaluated the effects of 8-hydroxydaidzein (8HD, an isoflavone isolated from fermented soy germ koji, and epirubicin (Epi, an antineoplastic agent, on the production of reactive oxygen species (ROS. We subsequently correlated the ROS levels to the anticancer mechanisms of Epi and 8HD in human colon adenocarcinoma Caco-2 cells. 8HD enhanced cytotoxicity of Epi and generated a synergistic effect. Epi and/or 8HD treatments increased the hydrogen peroxide and superoxide levels. Combined treatment markedly decreased mRNA expression levels of multidrug resistance protein 1 (MDR1, MDR-associated protein (MRP 1, and MRP2. 8HD significantly intensified Epi intracellular accumulation in Caco-2 cells. 8HD and/or Epi-induced apoptosis, as indicated by the reduced mitochondrial membrane potential and increased sub-G1 phase in cell cycle. Moreover, 8HD and Epi significantly enhanced the mRNA expressions of Bax, p53, caspases-3, -8, and -9. To our best knowledge, this study verifies for the first time that 8HD effectively circumvents MDR in Caco-2 cells through the ROS-dependent inhibition of efflux transporters and p53-mediated activation of both death receptor and mitochondrial pathways of apoptosis. Our findings of 8HD shed light on the future search for potential biotransformed isoflavones to intensify the cytotoxicity of anticancer drugs through simultaneous reversal of pump and nonpump resistance.

  3. Effect of Chum Salmon Egg Lectin on Tight Junctions in Caco-2 Cell Monolayers

    Directory of Open Access Journals (Sweden)

    Ryo Nemoto

    2015-05-01

    Full Text Available The effect of a chum salmon egg lectin (CSL3 on tight junction (TJ of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of β-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized β-actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine.

  4. Effect of chum salmon egg lectin on tight junctions in Caco-2 cell monolayers.

    Science.gov (United States)

    Nemoto, Ryo; Yamamoto, Shintaro; Ogawa, Tomohisa; Naude, Ryno; Muramoto, Koji

    2015-01-01

    The effect of a chum salmon egg lectin (CSL3) on tight junction (TJ) of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER) value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of β-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized β-actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine. PMID:25951005

  5. Rosa canina Extracts Have Antiproliferative and Antioxidant Effects on Caco-2 Human Colon Cancer

    Science.gov (United States)

    Jiménez, Sandra; Gascón, Sonia; Luquin, Asunción; Laguna, Mariano; Ancin-Azpilicueta, Carmen; Rodríguez-Yoldi, María Jesús

    2016-01-01

    The in vitro antiproliferative and antioxidant effects of different fractions of Rosa canina hips on human colon cancer cell lines (Caco-2) was studied. The compounds tested were total extract (fraction 1), vitamin C (fraction 2), neutral polyphenols (fraction 3) and acidic polyphenols (fraction 4). All the extracts showed high cytotoxicity after 72 h, both low and high concentrations. The flow cytometric analysis revealed that all the fractions produce disturbances in the cell cycle resulting in a concomitant cell death by an apoptotic pathway. Changes in the redox status of Caco-2 cells in response to Rosa canina hips were determined. Cells were exposed to hydrogen peroxide in presence of plant fractions and the production of Reactive Oxygen Species (ROS) was significantly decreased. Therefore, our data demonstrate that rosehip extracts are a powerful antioxidant that produces an antiproliferative effect in Caco-2 cells. Therefore, these results predict a promising future for Rosa canina as a therapeutic agent. Thus, this natural plant could be an effective component of functional foods addressed towards colorectal carcinoma. PMID:27467555

  6. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    Energy Technology Data Exchange (ETDEWEB)

    Arana, Maite Rocío, E-mail: arana@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Tocchetti, Guillermo Nicolás, E-mail: gtocchetti@live.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Domizi, Pablo, E-mail: domizi@ibr-conicet.gov.ar [Instituto de Biología Molecular y Celular de Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Arias, Agostina, E-mail: agoarias@yahoo.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo, E-mail: jprigalli@gmail.com [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Ruiz, María Laura, E-mail: ruiz@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); and others

    2015-09-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  7. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    International Nuclear Information System (INIS)

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  8. Nonpathogenic Escherichia coli Strain Nissle 1917 Inhibits Signal Transduction in Intestinal Epithelial Cells▿

    OpenAIRE

    Kamada, Nobuhiko; Maeda, Kenichi; Inoue, Nagamu; Hisamatsu, Tadakazu; Okamoto, Susumu; Hong, Kyong Su; Yamada, Takaya; Watanabe, Noriaki; Tsuchimoto, Kanji; Ogata, Haruhiko; Hibi, Toshifumi

    2007-01-01

    Although the probiotic Escherichia coli strain Nissle 1917 has been used for the treatment of inflammatory bowel diseases, the precise mechanisms of action of this strain remain unclear. In the present study, we estimated the anti-inflammatory effect of E. coli Nissle 1917 on inflammatory responses in vitro to determine the suppressive mechanism of Nissle 1917 on the inflammatory process. To determine the effect of E. coli Nissle 1917, the human colonic epithelial cell line HCT15 was incubate...

  9. Bacteria regulate intestinal epithelial cell differentiation factors both in vitro and in vivo

    OpenAIRE

    Becker, Svetlana; Oelschlaeger, Tobias A; Wullaert, Andy; Pasparakis, Manolis; Wehkamp, Jan; Stange, Eduard F; Gersemann, Michael

    2013-01-01

    Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. Methods...

  10. Bacteria Regulate Intestinal Epithelial Cell Differentiation Factors Both In Vitro and In Vivo

    OpenAIRE

    Becker, Svetlana; Oelschlaeger, Tobias A; Wullaert, Andy; Pasparakis, Manolis; Wehkamp, Jan; Stange, Eduard F; Gersemann, Michael

    2016-01-01

    Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. Method...

  11. Effects of hydrogen-rich medium on lipopolysaccharide-induced intestinal epithelial barrier dysfunction of human colon carcinoma cells%富氢液对脂多糖刺激离体肠上皮 屏障功能障碍的影响

    Institute of Scientific and Technical Information of China (English)

    杨涛; 谢克亮; 陈红光; 张红涛; 于洋; 王国林; 于泳浩

    2016-01-01

    Objective To investigate the effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction of human intestinal epithelial (Caco2) cells. Methods Caco2 cells (passages 28-35) were purchased from the Cell Bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences in Shanghai, China, and they were cultured in Dulbecco minimum essential medium (DMEM) containing 20% fetal bovine serum. These cells were randomly divided into four groups: control group (group A), hydrogen-rich medium group (group B), LPS group (group C) and LPS + hydrogen-rich medium group (group D). Cells were cultured with normal medium in group A and group C or with hydrogen-rich medium in group B and group D. Meanwhile, 1 g/L LPS was simultaneously added into group C and group D, while an equivalent volume of normal saline was added into group A and group B instead. In vitro intestinal epithelial models were reproduced with monolayer filter-grown Caco2 and intestinal epithelium. The trans-epithelial electrical resistance (TEER) in models of each group was measured at different incubation times (0, 3, 6, 12, 24 and 48 hours). Cell viability and cytotoxicity were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assay, respectively, after incubation for 24 hours. The expression levels of claudin-1 and occludin were respectively determined at 6, 12 and 24 hours of incubation by Western Blot assay. The morphological structure of claudin-1 and occludin was respectively observed after incubation for 24 hours with immunofluorescence staining. Results There was no statistical significance in variables between group A and group B. Compared with group A, it was shown that TEER was time-dependently decreased in groups C and D after 6 hours. Compared with group C, TEER in group D was increased after 6 hours. Compared with group A, the cell viability was significantly

  12. High hydrostatic pressure pre-treatment of whey proteins enhances whey protein hydrolysate inhibition of oxidative stress and IL-8 secretion in intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    André F. Piccolomini

    2012-06-01

    Full Text Available Background: High hyperbaric pressure treatment of whey protein isolate (WPI causes changes in the protein structure that enhances the anti-oxidant and anti-inflammatory effects of WPI. Objective: The aim of this study was to compare the anti-oxidant and anti-inflammatory effects of pressurized whey protein isolate (pWPI vs. native WPI (nWPI hydrolysates in Caco-2 cells exposed to hydrogen peroxide (H2O2. Design: Cells were cultured with different concentrations of pWPI or nWPI hydrolysates either 1 h before or 1 h after H2O2. Cell viability, IL-8 secretion, intracellular reactive oxygen species (ROS, and the medium anti-oxidant capacity (FRAP assay were measured. Results: Prior to and after H2O2 exposure, pWPI and nWPI hydrolysates inhibited IL-8 secretion and ROS generation, and increased FRAP activity in a dose-dependent manner. The maximal inhibition of H2O2-induced IL-8 secretion was greater with 2000 µg mL−1 of pWPI (50% vs. nWPI (30% hydrolysates. At the latter concentration, inhibition of H2O2-induced ROS formation reached 76% for pWPI, which was greater than for nWPI hydrolysates (32.5%. Conclusion: These results suggest that WPI hydrolysates can alleviate inflammation and oxidative stress in intestinal cells exposed to oxidative injury, which is further enhanced by hyperbaric pressure pre-treatment of WPI.

  13. In vitro small intestinal epithelial cell growth on a nanocomposite polycaprolactone scaffold

    OpenAIRE

    Gupta, Ashish; Vara, Dina S.; Punshon, Geoffrey; Sales, Kevin M.; Winslet, Marc C.; Seifalian, Alexander M.

    2009-01-01

    Tissue engineering of the small intestine remains experimental despite worldwide attempts to develop a functional substitute for short bowel syndrome. Most published studies have reported predominant use of PLLA (poly-L-lactide acid)/PGA (polyglycolic acid) copolymer as the scaffold material, and studies have been limited by in vivo experiments. This lack of progress has inspired a fresh perspective and provoked further investigation and development in this field of tissue engineering. In the...

  14. HYDROGEN-RICH MEDIUM AMELIORATES LIPOPOLYSACCHARIDE-INDUCED BARRIER DYSFUNCTION VIA RHOA-MDIA1 SIGNALING IN CACO-2 CELLS

    Science.gov (United States)

    Yang, Tao; Wang, Lu; Sun, Ruiqiang; Chen, Hongguang; Zhang, Hongtao; Yu, Yang; Wang, Yanyan; Wang, Guolin; Yu, Yonghao; Xie, Keliang

    2016-01-01

    ABSTRACT Gastrointestinal barrier dysfunction is associated with the severity and prognosis of sepsis. Hydrogen gas (H2) can ameliorate multiple organ damage in septic animals. Ras homolog gene family member A (RhoA) and mammalian diaphanous-related formin 1 (mDia1) are important to regulate tight junction (TJ) and adherens junction (AJ), both of which determine the integrity of the intestinal barrier. This study was aimed to investigate whether H2 could modulate lipopolysaccharide (LPS)-stimulated dysfunction of the intestinal barrier and whether RhoA-mDia1 signaling is involved. Caco-2 cells were exposed to different concentrations of LPS (1 μg/mL–1 mg/mL). The permeability of the intestinal barrier was evaluated by transepithelial resistance (TER) and fluorescein-isothiocyanate-dextran flux. Expression and distribution of occludin and E-cadherin were analyzed by Western blot and immunofluorescence. RhoA activity was measured by G-Lisa assay, and mDia1 expression was assessed by Western blot. LPS (100 μg/mL) decreased TER and increased fluorescein-isothiocyanate-dextran flux, which were alleviated by H2-rich medium. Also, H2 down-regulated LPS-induced oxidative stress. Moreover, H2 improved the down-regulated expression and redistribution of occludin and E-cadherin caused by LPS. Additionally, H2 alleviated LPS-caused RhoA activation, and the beneficial effects of H2 on barrier were counteracted by RhoA agonist CN03. Rho inhibitor C3 exoenzyme mitigated LPS-induced barrier breakdown. Furthermore, H2-rich medium increased mDia1 expression, and mDia1 knockdown abolished protections of H2 on barrier permeability. mDia1 knockdown eliminated H2-induced benefits for occludin and E-cadherin. These findings suggest that H2 improves LPS-induced hyperpermeability of the intestinal barrier and disruptions of TJ and AJ by moderating RhoA-mDia1 signaling. PMID:26529665

  15. Autophagy Deficiency Diminishes Indomethacin-Induced Intestinal Epithelial Cell Damage through Activation of the ERK/Nrf2/HO-1 Pathway.

    Science.gov (United States)

    Harada, Satoshi; Nakagawa, Takatoshi; Yokoe, Shunichi; Edogawa, Shoko; Takeuchi, Toshihisa; Inoue, Takuya; Higuchi, Kazuhide; Asahi, Michio

    2015-12-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) can cause epithelial cell damage in the stomach, intestine, and colon. NSAIDs are reported to induce autophagy and apoptosis in intestinal epithelial cells; however, their role in cell damage is poorly understood. To examine the role of autophagy in cell damage, we used autophagy-related gene Atg5-conditional knockout mice, in which the Atg5 gene is only knocked out in intestinal epithelial cells. In an indomethacin (IM)-induced gastrointestinal ulcer mouse model, intestinal epithelium damage was reduced in Atg5-conditional knockout mice compared with wild-type mice. IM-induced damage in IEC6 rat intestinal epithelial cells was reduced when Atg5 was silenced (IEC6shAtg5 cells). Western blot analyses indicated that IM-induced apoptosis decreased, and the potent, oxidative stress-related extracellular signal-regulated kinase (ERK)/nuclear factor-erythroid2-like2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway was upregulated in IEC6shAtg5 cells. An experiment using a reactive oxygen species (ROS)-sensitive fluorescent dye in IEC6shAtg5 cells revealed that the amount of ROS at the baseline and the rate of increase after IM treatment were lower than in intact IEC6 cells. The mitochondrial membrane potential at the baseline and the reduction rate in IM-treated IEC6shAtg5 cells were lower than in intact IEC6 cells, indicating that autophagy deficiency increased ROS production caused by mitochondrial disturbance. Furthermore, MnTMPyP, a manganese-superoxide dismutase mimetic, significantly inhibited IM-induced autophagy and subsequent apoptosis as well as activation of the ERK/Nrf2/HO-1 pathway. These data suggest that autophagy deficiency and subsequent activation of the ERK/Nrf2/HO-1 pathway diminished IM-induced, apoptosis-mediated intestinal epithelial cell damage, and genetic analyses of single nucleotide polymorphisms in autophagy-related genes could predict NSAID-induced intestinal injury. PMID:26404472

  16. Challenges of culturing human norovirus in three-dimensional organoid intestinal cell culture models.

    Directory of Open Access Journals (Sweden)

    Efstathia Papafragkou

    Full Text Available Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407 or human epithelial colorectal adenocarcinoma cells (Caco-2 growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin. Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8. At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

  17. Effects of quinoa hull meal on piglet performance and intestinal epithelial physiology

    DEFF Research Database (Denmark)

    Carlson, Dorthe; Fernández, José Adalberto; Poulsen, Hanne Damgaard;

    2012-01-01

    Saponin-containing feed additives have shown positive effects on pig performance. Quinoa hull has high saponin content and may be of interest as a feed additive. This study aimed to evaluate quinoa hull meal (QHM) as a feed additive in a pig diet. The effects of QHM were assessed for three dosages...... of South American (SA) origin (100, 300 and 500 mg/kg) and one dosage of Danish (DK) quinoa (300 mg/kg). In addition, the effect of dietary SA-QHM and SA-QHM-extract on jejunal epithelial physiology was studied ex vivo in Ussing chambers. The experiment included 400 piglets weaned at 28 ± 2 days of...

  18. Effect of Ozone on Intestinal Epithelial Homeostasis in a Rat Model

    Directory of Open Access Journals (Sweden)

    Igor Sukhotnik

    2015-01-01

    Full Text Available Background: The positive effects of ozone therapy have been described in many gastrointestinal disorders. The mechanisms of this positive effect of ozone therapy are poorly understood. The purpose of the present study was to investigate whether the use of ozone may potentiate the gut intestinal mucosal homeostasis in a rat model. Methods: Adult rats weighing 250–280 g were randomly assigned to one of three experimental groups of 8 rats each: 1 Control rats were given 2 mL of water by gavage and intraperitoneally (IP for 5 days; 2 O3-PO rats were treated with 2 mL of ozone/oxygen mixture by gavage and 2 mL of water IP for 5 days; 3 O3-IP rats were treated with 2 mL of water by gavage and 2 mL of ozone/oxygen mixture IP for 5 days. Rats were sacrificed on day 6. Bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth, and cell proliferation and apoptosis were evaluated following sacrifice. Results: The group of O3-IP rats demonstrated a greater jejunal and ileal villus height and crypt depth, a greater enterocyte proliferation index in jejunum, and lower enterocyte apoptosis in ileum compared to control animals. Oral administration of the ozone/oxygen mixture resulted in a less significant effect on cell turnover. Conclusions: Treatment with an ozone/oxygen mixture stimulates intestinal cell turnover in a rat model. Intraperitoneal administration of ozone resulted in a more significant intestinal trophic effect than oral administration.

  19. Effect of Ozone on Intestinal Epithelial Homeostasis in a Rat Model

    Science.gov (United States)

    Sukhotnik, Igor; Starikov, Alona; Coran, Arnold G.; Pollak, Yulia; Sohotnik, Rima; Shaoul, Ron

    2015-01-01

    Background: The positive effects of ozone therapy have been described in many gastrointestinal disorders. The mechanisms of this positive effect of ozone therapy are poorly understood. The purpose of the present study was to investigate whether the use of ozone may potentiate the gut intestinal mucosal homeostasis in a rat model. Methods: Adult rats weighing 250–280 g were randomly assigned to one of three experimental groups of 8 rats each: 1) Control rats were given 2 mL of water by gavage and intraperitoneally (IP) for 5 days; 2) O3-PO rats were treated with 2 mL of ozone/oxygen mixture by gavage and 2 mL of water IP for 5 days; 3) O3-IP rats were treated with 2 mL of water by gavage and 2 mL of ozone/oxygen mixture IP for 5 days. Rats were sacrificed on day 6. Bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth, and cell proliferation and apoptosis were evaluated following sacrifice. Results: The group of O3-IP rats demonstrated a greater jejunal and ileal villus height and crypt depth, a greater enterocyte proliferation index in jejunum, and lower enterocyte apoptosis in ileum compared to control animals. Oral administration of the ozone/oxygen mixture resulted in a less significant effect on cell turnover. Conclusions: Treatment with an ozone/oxygen mixture stimulates intestinal cell turnover in a rat model. Intraperitoneal administration of ozone resulted in a more significant intestinal trophic effect than oral administration. PMID:25717388

  20. Strategies for Profiling Single Mouse Intestinal Epithelial Cells by Targeted Gene Expression

    OpenAIRE

    McDowell, W.; Box, A. (Antonio); Staehling, K.; Wang, F.; Li, L.; Zueckert-Gaudenz, K.

    2014-01-01

    Targeted gene expression profiling of single cells permits the study of heterogeneity in cell populations. Here, a pool of mouse intestinal crypt-base CD44+/GRP78- cells was collected by fluorescence activated cell sorting. Aliquots were either loaded onto Fluidigm's C1 System for microfluidic cell capture and cDNA synthesis in nanoliter volumes, or flow-sorted directly into individual PCR plate wells for cDNA synthesis in microliter volumes. The pre-amplified cDNAs were transferred to the Bi...

  1. Immunomodulatory Effects of Lactobacillus plantarum Lp62 on Intestinal Epithelial and Mononuclear Cells

    Science.gov (United States)

    Alves Melo, Tauá; Almeida, Milena Evangelista; Passos Rezende, Rachel

    2016-01-01

    Probiotic lactic acid bacteria are known for their ability to modulate the immune system. They have been shown to inhibit inflammation in experiments with animal models, cell culture, and clinical trials. The objective of this study was to elucidate the anti-inflammatory potential of Lactobacillus plantarum Lp62, isolated from cocoa fermentation, in a cell culture model. Lp62 inhibited IL-8 production by Salmonella Typhi-stimulated HT-29 cells and prevented the adhesion of pathogens to these epithelial cells. The probiotic strain was able to modulate TNF-α, IL1-β, and IL-17 secretion by J774 macrophages. J774 activation was reduced by coincubation with Lp62. PBMC culture showed significantly higher levels of CD4+CD25+ T lymphocytes following treatment with Lp62. Probiotics also induced increased IL-10 secretion by mononuclear cells. L. plantarum Lp62 was able to inhibit inflammatory stimulation in epithelial cells and macrophages and activated a tolerogenic profile in mononuclear cells of healthy donors. These results indicate this strain for a possible application in the treatment or prevention of inflammatory diseases. PMID:27446958

  2. Effect of WPI Maillard reaction on the Caco-2 cells inhibition%乳清蛋白Maillard反应产物对Caco-2细胞抗增殖抑制的影响

    Institute of Scientific and Technical Information of China (English)

    杨晶; 张凤阳; 田雨; 田然; 姜瞻梅

    2013-01-01

    Effects of Maillard reaction products (MRPs) of whey protein isolate (WPI) and reducing sugar (ribose,galactose and lactose) on growth inhibition of Caco-2 cells were studied by Caco-2 cells culture technology combined with MTT assay,in order to evaluate the toxicology characteristics of WPI-sugar MRPs.It was shown that WPI-sugar MRPs prepared after the 0,2,4 h heat treatment made relative growth rate of Caco-2 cells reduced with the concentration of WPI-sugar MRPs increased.Under the same concentration of WPI-sugar MRPs,effects ofWPI-sugar MRPs prepared after different heat treatment on relative growth rate of Caco-2 cells were significantly different.The longer the heating time ofWPI-sugar MRPs,the lower relative growth rate of Caco-2 cells.The relative growth rate of Caco-2 cells of WPI,WPI-ribose,WPI-galactose and WPI-lactose MRPs prepared after the heating time of 4 hours,was 68.7%,87.5%,86.8% and 70.9%,respectively.This indicates that WPI-sugar MRPs can slightly inhibit Caco-2 cells to grow,and they can be widely used in food industry as functional ingredients.%利用Caco-2细胞培养技术,结合MTT检测法,研究乳清分离蛋白(WPI)和还原糖(核糖、半乳糖以及乳糖)的美拉德(Maillard)反应产物对Caco-2细胞生长抑制的影响,以判断乳清蛋白Maillard反应产物的毒理学特性.结果表明,加热处理时间为0,2和4h的乳清蛋白Maillard反应产物,在作用Caco-2细胞质量浓度在0.01~2 g/L范围内,随着Maillard反应产物浓度的增加,Caco-2细胞存活率降低.在相同的作用质量浓度条件下,不同加热处理时间的乳清蛋白Maillard反应产物对Caco-2细胞存活率影响差异显著,即加热处理时间越长的乳清蛋白Maillard反应产物,对Caco-2细胞抑制作用越大.加热处理时间为4h的WPI、WPI-核糖、WPI-半乳糖和WPI-乳糖的Maillard反应产物对Caco-2细胞存活率分别为68.7%,87.5%,86.8%和70.9%,这说明乳清蛋白Maillard反应产物对Caco

  3. Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.

    Science.gov (United States)

    Donowitz, M; Janecki, A; Akhter, S; Cavet, M E; Sanchez, F; Lamprecht, G; Zizak, M; Kwon, W L; Khurana, S; Yun, C H; Tse, C M

    2000-01-01

    NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number. PMID:11193592

  4. TO901317 regulating apolipoprotein M expression mediates via the farnesoid X receptor pathway in Caco-2 cells

    OpenAIRE

    Berggren-Söderlund Maria; Shi Yuanping; Wei Jiang; Wang Zongchun; Luo Guanghua; Zhang Xiaoying; Di Dongmei; Zhu Chunhua; Nilsson-Ehle Peter; Xu Ning

    2011-01-01

    Abstract Background Apolipoprotein M (apoM) may have potential antiatherosclerotic properties. It has been reported that apoM expression could be regulated by many intracellar and extracellar factors. In the present study we further investigated regulation of apoM expression in Caco-2 cells stimulated by a liver X receptor (LXR) agonist, TO901317. Materials and methods Caco-2 cells were cultured in the presence of either TO901317, farnesoid X receptor (FXR) antagonist guggulsterone or TO90131...

  5. Flagellin-induced tolerance of the Toll-like receptor 5 signaling pathway in polarized intestinal epithelial cells.

    Science.gov (United States)

    Sun, Jun; Fegan, Pamela E; Desai, Anjali S; Madara, James L; Hobert, Michael E

    2007-03-01

    Salmonella typhimurium is a gram-negative enteric pathogen that invades the mucosal epithelium and is associated with diarrheal illness in humans. Flagellin from S. typhimurium and other gram-negative bacteria has been shown to be the predominant proinflammatory mediator through activation of the basolateral Toll-like receptor 5 (TLR5). Recent evidence has shown that prior exposure can render immune cells tolerant to subsequent challenges by TLR ligands. Accordingly, we examined whether prior exposure to purified flagellin would render human intestinal epithelial cells insensitive to future contact. We found that flagellin-induced tolerance is common to polarized epithelial cells and prevents further activation of proinflammatory signaling cascades by both purified flagellin and Salmonella bacteria but does not affect TNF-alpha stimulation of the same pathways. Flagellin tolerance is a rapid process that does not require protein synthesis, and that occurs within 1 to 2 h of flagellin exposure. Prolonged flagellin exposure blocks activation of the NF-kappaB, MAPK, and phosphoinositol 3-kinase signaling pathways and results in the internalization of a fraction of the basolateral TLR5 without affecting the polarity or total expression of TLR5. After removal of flagellin, cells require more than 24 h to fully recover their ability to mount a normal proinflammatory response. We have found that activation of phosphoinositol 3-kinase and Akt by flagellin has a small damping effect in the early stages of flagellin signaling but is not responsible for tolerance. Our study indicates that inhibition of TLR5-associated IL-1 receptor-associated kinase-4 activity occurs during the development of flagellin tolerance and is likely to be the cause of tolerance. PMID:17138965

  6. Oat β-glucan depresses SGLT1- and GLUT2-mediated glucose transport in intestinal epithelial cells (IEC-6).

    Science.gov (United States)

    Abbasi, Nazanin N; Purslow, Peter P; Tosh, Susan M; Bakovic, Marica

    2016-06-01

    Oat β-glucan consumption is linked to reduced risk factors associated with diabetes and obesity by lowering glycemic response and serum level of low-density lipoproteins. The purpose of this study was to identify the mechanism of action of oat β-glucan at the interface between the gut wall and the lumen responsible for attenuating glucose levels. We proposed that viscous oat β-glucan acts as a physical barrier to glucose uptake in normally absorptive gut epithelial cells IEC-6 by affecting the expression of intestinal glucose transporters. Concentration and time-dependent changes in glucose uptake were established by using a nonmetabolizable glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose. The effectiveness of nutrient transport in IEC-6 cells was shown by significant differences in glucose uptake and corresponding transporter expression. The expressions of glucose transporters sodium-glucose-linked transport protein 1 (SGLT1) and glucose transporter 2 (GLUT2) increased with time (0-60 minutes) and glucose levels (5-25 mmol/L). The suppression of glucose uptake and SGLT1 and GLUT2 expression by increasing concentrations (4-8 mg/mL) of oat β-glucan demonstrated a direct effect of the physical properties of oat β-glucan on glucose transport. These results affirmed oat β-glucan as a dietary agent for minimizing postprandial glucose and showed that modulating the activity of the key intestinal glucose transporters with oat β-glucan could be an effective way of lowering blood glucose levels in patients with diabetes. PMID:27188900

  7. Differential Effect of Lactobacillus johnsonii BFE 6128 on Expression of Genes Related to TLR Pathways and Innate Immunity in Intestinal Epithelial Cells.

    Science.gov (United States)

    Seifert, Stephanie; Rodriguez Gómez, Manuel; Watzl, Bernhard; Holzapfel, Wilhelm H; Franz, Charles M A P; Vizoso Pinto, María G

    2010-12-01

    Probiotics have been shown to enhance immune defenses, but their mechanisms of action are only partially understood. We investigated the modulation of signal pathways involved in innate immunity in enterocytes by Lactobacillus johnsonii BFE 6128 isolated from 'Kule naoto', a Maasai traditional fermented milk product. This lactobacillus sensitized HT29 intestinal epithelial cells toward recognition of Salmonella enterica serovar Typhimurium by increasing the IL-8 levels released after challenge with this pathogen and by differentially modulating genes related to toll-like receptor (TLR) pathways and innate immunity. Thus, the modulation of pro-inflammatory mediators and TLR-pathway-related molecules may be an important mechanism contributing to the potential stimulation of innate immunity by lactobacilli at the intestinal epithelial level. PMID:26781315

  8. Probiotic modulation of dendritic cells co-cultured with intestinal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Ji Yeun Kim; Myeong Soo Park; Geun Eog Ji

    2012-01-01

    AIM:TO investigate cytokine production and cell surface phenotypes of dendritic cells (DC) in the presence of epithelial cells stimulated by probiotics.METHODS:Mouse DC were cultured alone or together with mouse epithelial cell monolayers in normal or inverted systems and were stimulated with heat-killed probiotic bacteria,Bifidobacteriumlactis AD011 (BL),Bifidobacterium bifidum BGN4 (BB),Lactobacillus casei IBS041 (LC),and Lactobacillus acidophilus AD031 (LA),for 12 h.Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent assay and phenotypic analysis of DC was investigated by flow cytometry.RESULTS:BB and LC in single-cultured DC increased the expression of I-Ad,CD86 and CD40 (I-Ad,18.51 vs 30.88,46.11; CD86,62.74 vs 92.7,104.12; CD40,0.67vs 6.39,3.37,P < 0.05).All of the experimental probiotics increased the production of inflammatory cytokines,interleukin (IL)-6 and tumor necrosis factor (TNF)-α.However,in the normal co-culture systems,LC and LA decreased the expression of I-Ad (39.46 vs 30.32,33.26,P < 0.05),and none of the experimental probiotics increased the levels of IL-6 or TNF-α.In the inverted coculture systems,LC decreased the expression of CD40 (1.36 vs-2.27,P < 0.05),and all of the experimental probiotics decreased the levels of IL-6.In addition,BL increased the production of IL-10 (103.8 vs 166.0,P< 0.05) and LC and LA increased transforming growth factor-3 secretion (235.9 vs 618.9,607.6,P < 0.05).CONCLUSION:These results suggest that specific probiotic strains exert differential immune modulation mediated by the interaction of dendritic cells and epithelial cells in the homeostasis of gastrointestinal tract.

  9. Prune extract (Prunus domestica L.) suppresses the proliferation and induces the apoptosis of human colon carcinoma Caco-2.

    Science.gov (United States)

    Fujii, Takashi; Ikami, Takao; Xu, Jin-Wen; Ikeda, Katsumi

    2006-10-01

    Prunes are the dried fruits of certain cultivars of Prunus domestica L., and are recognized as a health food. The separated ethanol fraction from concentrated prune juice by DIAION HP-20 (PE) was investigated for cytotoxic effects on two different cancer cell lines in vitro. PE dose-dependently reduced the viable cell number of Caco-2, KATO III, but does not reduce the viable cell number of human normal colon fibroblast cells (CCD-18Co) used as a normal cell model. PE treatment for 24 h led to apoptotic changes in Caco-2 such as cell shrinkage and blebbed surfaces due to the convolutions of nuclear and plasma membranes and chromatin condensation, but this was not observed in CCD-18Co. PE induced nucleosomal DNA fragmentation typical of apoptosis in Caco-2 after 24 h of treatment. These results show that PE induced apoptosis in Caco-2. Furthermore, by Caco-2 treatment with H2O2 chelator catalase and Ca2+-chelator BAPTA/AM, the PE-induced cytotoxic pathway was completely blocked, and the viable cell number of Caco-2 was not affected. PMID:17190111

  10. Butyrate attenuates lipopolysaccharide-induced inflammation in intestinal cells and Crohn's mucosa through modulation of antioxidant defense machinery.

    Science.gov (United States)

    Russo, Ilaria; Luciani, Alessandro; De Cicco, Paola; Troncone, Edoardo; Ciacci, Carolina

    2012-01-01

    Oxidative stress plays an important role in the pathogenesis of inflammatory bowel disease (IBD), including Crohn's disease (CrD). High levels of Reactive Oxygen Species (ROS) induce the activation of the redox-sensitive nuclear transcription factor kappa-B (NF-κB), which in turn triggers the inflammatory mediators. Butyrate decreases pro-inflammatory cytokine expression by the lamina propria mononuclear cells in CrD patients via inhibition of NF-κB activation, but how it reduces inflammation is still unclear. We suggest that butyrate controls ROS mediated NF-κB activation and thus mucosal inflammation in intestinal epithelial cells and in CrD colonic mucosa by triggering intracellular antioxidant defense systems. Intestinal epithelial Caco-2 cells and colonic mucosa from 14 patients with CrD and 12 controls were challenged with or without lipopolysaccaride from Escherichia coli (EC-LPS) in presence or absence of butyrate for 4 and 24 h. The effects of butyrate on oxidative stress, p42/44 MAP kinase phosphorylation, p65-NF-κB activation and mucosal inflammation were investigated by real time PCR, western blot and confocal microscopy. Our results suggest that EC-LPS challenge induces a decrease in Gluthation-S-Transferase-alpha (GSTA1/A2) mRNA levels, protein expression and catalytic activity; enhanced levels of ROS induced by EC-LPS challenge mediates p65-NF-κB activation and inflammatory response in Caco-2 cells and in CrD colonic mucosa. Furthermore butyrate treatment was seen to restore GSTA1/A2 mRNA levels, protein expression and catalytic activity and to control NF-κB activation, COX-2, ICAM-1 and the release of pro-inflammatory cytokine. In conclusion, butyrate rescues the redox machinery and controls the intracellular ROS balance thus switching off EC-LPS induced inflammatory response in intestinal epithelial cells and in CrD colonic mucosa. PMID:22412931

  11. The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Mediating Interactions with Intestinal Epithelial Cells and Chemokine Induction▿

    OpenAIRE

    Hafez, Mohamed; Hayes, Kelly; Goldrick, Marie; Warhurst, Geoff; Grencis, Richard; Roberts, Ian S

    2009-01-01

    Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. coli. In this paper, we investigate the role of the K5 capsule in mediating interactions between Nissle 1917 and intestinal epithelial cells. We show that the loss of capsule significantly reduced ...

  12. Enhanced Wound Healing by Recombinant Escherichia coli Nissle 1917 via Human Epidermal Growth Factor Receptor in Human Intestinal Epithelial Cells: Therapeutic Implication Using Recombinant Probiotics

    OpenAIRE

    Choi, Hye Jin; Ahn, Jung Hoon; Park, Seong-Hwan; Do, Kee Hun; Kim, Juil; Moon, Yuseok

    2012-01-01

    The gastrointestinal mucosa has a remarkable ability to repair damage with the support of epidermal growth factor (EGF), which stimulates epithelial migration and proliferative reepithelialization. For the treatment of mucosal injuries, it is important to develop efficient methods for the localized delivery of mucoactive biotherapeutics. The basic idea in the present study came from the assumption that an intestinal probiotic vehicle can carry and deliver key recombinant medicinal proteins to...

  13. 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

    OpenAIRE

    Nzakizwanayo, Jonathan; Kumar, Sandeep; Ogilvie, Lesley A.; Patel, Bhavik A; Dedi, Cinzia; Wendy M. Macfarlane; Jones, Brian V.

    2015-01-01

    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 t...

  14. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Kazutoyo Yoda

    2012-06-01

    Full Text Available Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.

  15. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells

    OpenAIRE

    Yoda, Kazutoyo; He, Fang; Miyazawa, Kenji; Hiramatsu, Masaru

    2012-01-01

    Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.Key...

  16. Ectopic splenic tissues mimicking gastro-intestinal stromal tumour in a patient after splenectomy for a giant epithelial cyst of spleen: A case report

    OpenAIRE

    Chung Kam Man; Lau Hiu Yan Stephanie; Lau Wan Yee

    2015-01-01

    Introduction: Ectopic splenic tissues left after a previous splenectomy can masquerade as a gastro-intestinal stromal tumour (GIST). Presentation of case: Splenectomy was carried out for a 17-year-old girl with a giant epithelial cyst of spleen. Four years later, an upper endoscopy carried out for dyspepsia revealed two sub-mucosal lesions at the posterior wall of the gastric fundus. Computed tomography diagnosed a GIST. At operation, a dump-bell shaped extragastric mass was excised. Histo...

  17. Inhibition of intestinal epithelial apoptosis improves survival in a murine model of radiation combined injury.

    Science.gov (United States)

    Jung, Enjae; Perrone, Erin E; Brahmamdan, Pavan; McDonough, Jacquelyn S; Leathersich, Ann M; Dominguez, Jessica A; Clark, Andrew T; Fox, Amy C; Dunne, W Michael; Hotchkiss, Richard S; Coopersmith, Craig M

    2013-01-01

    World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target. PMID:24204769

  18. Cholinergic muscarinic receptor activation augments murine intestinal epithelial cell proliferation and tumorigenesis

    International Nuclear Information System (INIS)

    Previously, we showed that M3 muscarinic receptor (M3R; gene name Chrm3) deficiency attenuates murine intestinal neoplasia, supporting the hypothesis that muscarinic receptors play an important role in intestinal tumorigenesis. To test this hypothesis, in the present study we treated mice with bethanechol, a non-selective muscarinic receptor agonist without nicotinic receptor activity, and examined its effects on azoxymethane (AOM)-induced colon neoplasia. Mice were provided with drinking water containing 400 μg/mL bethanechol chloride or water without additions (control) for a total of 20 weeks, a period that included the initial 6 weeks when mice received intraperitoneal injections of AOM. When euthanized at week 20, control mice had 8.0 ± 1.3 tumors per animal, whereas bethanechol-treated mice had 10.4 ± 1.5 tumors per mouse (mean ± SE; P = 0.023), a 30% increase. Strikingly, tumor volume per animal was increased 52% in bethanechol-treated compared with control mice (179.7 ± 21.0 vs. 111. 8 ± 22.4 mm3; P = 0.047). On histological examination, bethenechol-treated mice also had more adenocarcinomas per animal (8.0 ± 1.0 vs. 4.1 ± 0.6 for control mice, P = 0.0042). Cell proliferation in both normal mucosa and adenocarcinomas was increased in bethanechol-treated compared to control mice. Also, in tumors, bethanechol treatment increased expression of Chrm3, Egfr and post-Egfr signaling molecules Myc and cyclin D1. Bethanechol treatment increased the thickness of normal colonic mucosa and the expression of selected matrix metalloproteinase (Mmp) genes, including Mmp7, Mmp10 and Mmp13. These findings support a prominent role for muscarinic receptors in colon neoplasia, and identify post-receptor signaling molecules as potential therapeutic targets

  19. Inhibition of intestinal epithelial apoptosis improves survival in a murine model of radiation combined injury.

    Directory of Open Access Journals (Sweden)

    Enjae Jung

    Full Text Available World conditions place large populations at risk from ionizing radiation (IR from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA. While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01. Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01. These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.

  20. Biphasic regulation of P-glycoprotein function and expression by NO donors in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Ru DUAN; Nan HU; Hai-yan LIU; Jia LI; Hai-fang GUO; Can LIU; Li LIU; Xiao-dong LIU

    2012-01-01

    Aim:To investigate the effects of nitric oxide (NO) donors on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells.Methods:Caco-2 cells were exposed to NO donors for designated times.P-gp function and expression were assessed using Rhodamine123 uptake assay and Western blotting,respectively.Intracellular reactive oxygen species (iROS) and intracellular reactive nitrogen species (iRNS) levels were measured using ROS and RNS assay kits,respectively.Results:Exposure of Caco-2 cells to 0.1 or 2 mmol/L of sodium nitroprusside (SNP) affected the function and expression of P-gp in concentration- and time-dependent manners.A short-term (4 h) exposure reduced P-gp function and expression accompanied with significantly increased levels of iROS and iRNS.In contrast,a long-term (24 h) exposure stimulated the P-gp function and expression.The stimulatory effects of 2 mmol/L SNP was less profound as compared to those caused by 0.1 mmol/L SNP.The other NO donors SIN-1 and SNAP showed similar effects.Neither the NO scavenger PTIO (2 mmol/L) nor soluble guanylate cyclase inhibitor ODQ (50 μmol/L) reversed the SNP-induced alteration of P-gp function.On the other hand,free radical scavengers ascorbate,glutathione and uric acid (2 mmol/L for each),PKC inhibitor chelerythrine (5 μmol/L),PI3K/Akt inhibitor wortmannin (1 pmol/L) and p38 MAPK inhibitor SB203580 (10 μmol/L) reversed the upregulation of P-gp function by the long-term exposure to SNP,but these agents had no effect on the impaired P-gp function following the short-term exposure to SNP.Conclusion:NO donors time-dependently regulate P-gp function and expression in Caco-2 cells:short-term exposure impairs P-gp function and expression,whereas long-term exposure stimulates P-gp function and expression.The regulation occurs via a NO-independent mechanism.

  1. Transcytosis of Aminopeptidase N in caco-2 cells is mediated by a Non-cytoplasmic Signal

    DEFF Research Database (Denmark)

    Vogel, L K; Norén, Ove; Sjöström, H

    1995-01-01

    transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were...... transported to the basolateral plasma membrane in proportions equivalent to the wild type enzyme. This shows that no cytoplasmic basolateral sorting signal is involved in directing aminopeptidase N to the basolateral plasma membrane. Both the wild type and the tail-less aminopeptidase N were transcytosed from...

  2. Blockage of protease-activated receptor 1 ameliorates heat-stress induced intestinal high permeability and bacterial translocation.

    Science.gov (United States)

    Xu, Qiu-lin; Guo, Xiao-hua; Liu, Jing-xian; Chen, Bin; Liu, Zhi-feng; Su, Lei

    2015-04-01

    Accumulated evidences indicate intestinal lesions play an important role in the pathogenesis of heatstroke. However, the underlying mechanisms by which heat stress causes intestinal barrier dysfunction and bacterial translocation remain unclear. In this study, we investigated the role of protease-activated receptor 1 (PAR1) in heat stress-induced intestinal hyper-permeability and bacterial translocation. Intestinal permeability in heat stressed mouse was evaluated by determining plasma endotoxin concentration and urinal lactulose/mannitol (L/M) ratio with gastric administration of L/M solution. Venous blood, liver, spleen and mesenteric lymph node tissues were collected for bacterial load test. Real time PCR was used to determine ileum PAR1 mRNA expression. In vitro study, permeability was assessed by determining trans-epithelial electrical resistance (TEER) in human intestinal Caco-2 cell line. RWJ-58259, a selective antagonist of PAR1, was used both in vivo and in vitro studies. The results showed that heat stress could increase ileum PAR1 mRNA level, urinal L/M ratio, plasma endotoxin concentration and bacterial load in the blood, spleen and mesenteric lymph nodes. Blocking PAR1 with RWJ-58259 (10 mg/kg) pretreatment could significantly reduce heat stress-induced above changes, but have no role to PAR1 mRNA level. In Caco-2 cells, heat stress-induced high permeability could also be reduced by RWJ-58259 (5-20 µmol/L). In summary, our results demonstrated that PAR1 signaling pathway may play an important role in the heat stress-induced elevation of intestinal permeability, bacterial translocation and the occurrence of endotoxemia. PMID:25492552

  3. Epithelial morphogenesis in three-dimensional cell culture system

    OpenAIRE

    Liu, Mengfei; 刘梦菲

    2014-01-01

    In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispens