439 CORE 8,9 † brought to you by by you to brought Another group of 8 5,7 2011 Society of Chemical Industry c Wageningen University & Research Publications Research & University Wageningen by provided ˜ na-Calahorro [email protected] In contrast, capsaicin has shown to reduce 10 ´ ordoba, Cordoba, Spain. although a reduction in TEER is more difficult to interpret 12 11,12 Current address: Departamento de Bioquimica ydad Biologia de Molecular, C Universi- Correspondence to: Robert AM Vreeburg, Wageningen University andCentre, Research Food & BiobasedNetherlands. E-mail: Research, ram P.O. Box 17, 6700 AA Wageningen, The Wageningen University andWageningen, The Research Netherlands Centre, Food & Biobased Research, Caco-2 cells can resemble small intestinal epithelial cells ∗ † and serve ascompounds a on barrier model function. The systemfood fruit- compound and to quercetin vegetable-derived has study been shownepithelial effects to electrical increase resistance of the (TEER) trans- single over a food Caco-2 monolayer. the immune response duewould challenge to the immune passage system. of foreign peptides that since the reduction might alsotoxicity. be caused by cell damage due to TEER, The increased TEERclaudin4 was (cldn4) correlated , with andbroad-spectrum the higher stimulation abundance was kinase of inhibited inhibitors. by compounds that have been shown toin induce epithelial the resistance Caco-2(PUFAs), model which system wereepithelial are leakage. able polyunsaturated to fatty alleviate acids interleukin-4 induced 2 Claudins with the 3 1 ). Allergic reactions CLDN4 4–6 Esther E van Wezel, Francisco Oca ∗ A compromised intestinal barrier function 2 : 439–444 www.soci.org 92 2012; Changes in paracellular permeability are brought Tight junctions thus form an important cellular apple; Caco-2; claudin 4; permeability; resistance; TEER 2,3 2

2011 Society of Chemical Industry Tight junctions are complex protein assemblies in which c CONCLUSION: Crude apple extract inducesdetermine a whether these higher results paracellular can resistance be extrapolated in to differentiated human Caco-2 small cells. intestinal epithelia. Future research will Keywords: the three-dimensional structure andcomponents functionality are of just individual beginning to be unravelled. about by changes inmembrane phosphorylation, properties. protein composition and structure thatepithelium. determines Transport of compounds overall throughis the barrier driven tight junctions by passive properties diffusionand down the of selectivity an is electrochemical determined the gradient, by tight-junction composition. J Sci Food Agric The small intestinal epithelium formswith a the large outside interacting world. Its surface for prime energy and function biosynthesis, is combined tobarrier with absorb acting to nutrients as a protect selective composed the of human a monolayer system. whichinterspersed The consists with mainly epithelial of goblet barrier enterocytes cells is and endocrine cells, INTRODUCTION BACKGROUND: The small intestinal epithelium functions both toluminal, absorb nutrients, world and and to provide the a barrier humanis between body. the controlled outside, One by of the theintestinal properties passageways epithelium of across to tight study the the junction intestinal effect complexes. epithelium of crude is We apple paracellular used extracts diffusion, on aRESULTS: which paracellular differentiated permeability. Caco-2 Exposure monolayer of asdetermined a crude as model trans-epithelial apple for small electricalpresent. homogenate resistance The to TEER-enhancing (TEER). effect the Thisnot of inhibited increase differentiated apple by was protein extract Caco-2 kinase linearly was inhibitors.junction cells Apple-induced due related related resistance , to was to increased including accompanied factors claudin the by the 4 mainly increased ( concentration expression present paracellular of of in several resistance, tight apple the cortex, and the induction was Abstract cells Robert AM Vreeburg, and Jurriaan J Mes resistance and claudin 4 expression in Caco-2 (wileyonlinelibrary.com) DOI 10.1002/jsfa.4598 Apple extract induces increased epithelial Research Article Received: 26 April 2011 Revised: 8 July 2011 Accepted: 9 July 2011 Published online in Wiley Online Library: 3 October 2011 is suggested to be associated withof the pathogenesis intestinal of a diseases number and food allergies. are hypothesised to formand pores in in this thetransport. tight-junction way complex, control the nature and flux of paracellular to food productsincreased or paracellular permeability, food which in intolerance turn can can exacerbate be associated with paracellular spacejunctions. between cells being sealed off by tight View metadata, citation and similar papers at core.ac.uk at papers similar and citation metadata, View . - et al stocks digested × : 439–444 The effects 92 18 in vitro 2012; -tetraacetic acid  N ,  N , FBS was added to the KCl, with the respective N , + 1 N − L TriZol was followed with µ -hexahydro-1,4-diazepine hy- J Sci Food Agric H NaCl–5 mmol L In brief, TriZol (Invitrogen, Bleiswijk, The inhibitor; inhibitor only) was added to the 1 − + 15 . 15 . using either MS-Excel, or SPSS (PASW Statistics, FBS was added to the basal compartment. TEER et al 17 + et al Primer sequences and PCR settings were as described A subset of the total expression data was selected 15 16 FBS: 150 mmol L Reverse transcriptase quantitative polymerase chain reac- -tests, or with univariate analysis of variance with a Dunnett’s DNaseI (Sigma-Aldrich) treatment and RNeasy (Qiagen, Venlo,Netherlands) The clean-up. cDNA was synthesised with iScript (BioRad, Veenendaal, The Netherlands). Affymetrixwere used HG-U133 with microarrays GenChip hybridisation kits,apple using andgcRMA. controls. Microarray data was normalised using meter (Millipore, Molsheim, France), twice before sample addition. Medium was removed from theand apical diluted samples and were basal added compartments, fresh to DMEM the apical compartment and was determined after various times of exposure. Inhibitor studies Inhibitors [1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7); 1- (5-iodonaphthalene-1-sulfonyl)-1 as indicator. The cellexposure experiments. medium TEER was measured was using a replaced MilliCell-ERS the day before the drochloride(indol-3-yl)maleimide (ML-7); hydrochloride (GF 109 203X); staurosporine; and 3-[1-(dimethylaminopropyl)indol-3-yl]-4- 1,2-bis(2-aminophenoxy)ethane- IBM, Amsterdam, Thedetermined according Netherlands). to Benjamini False and Hochberg. discovery rate was RESULTS AND DISCUSSION Effect of apple extract on Caco-2 barrierCrude function apple extractselectrical resistance induced (TEER) anThe of effect increase occurred the inlittle within Caco-2 trans-epithelial further 1 monolayer h increase of (Fig.for in 1a). exposure, at TEER after least was which 6 observed h no (Fig. (data 1) or not and shown). lasted Induction of TEER values by Statistical analysis Statistical significance was calculatedt with individual, two-sided post-hoc test, inhibitor. After a 30 minbasal incubation, medium compartments from the was apical removed(apple and only; and apple the respectiveapical samples compartment and freshbasal compartment. DMEM expression studies RNA wasby extracted Vreeburg and cDNA was prepared as described by Vreeburg based on , and statistics were applied to this subset. tion (RT-qPCR) wastein P0 performed (RPLP0) and glyceraldehyde-3-phosphate using dehydrogenase (GAPDH) as ribosomal reference genes, phosphopro- whichpurpose. have been validated for this Netherlands) extraction with 200 of inhibitors were testedincrease by in comparing presence of the the apple-induced inhibitorincrease with TEER in the absence apple-induced of TEER the inhibitor. were prepared in DMSOmoved (96% from the ethanol apical compartment for and replaced with H-7). 1+ : Medium 1 DMEM was re- tetrakis(acetoxymethyl ester) (BAPTA-AM)] were purchasedSigma-Aldrich from (Zwijndrecht, The Netherlands) and 2000 1 C C. of − ◦ ◦ KCl KCl was 2 1 1 80 3 − − C. The at 4 − ◦ digested g www.soci.org RAM Vreeburg and grown × 2011 Society of Chemical Industry at 4 1 NaHCO − c of each). The g 1 KCl was added In this paper we − in vitro , the headspace 1 1 × 3 − digestion controls. − 14 cells L 9 C) fetal bovine serum NaCl–5 mmol L NaCl–5 mmol L ◦ 1 1 10 FBS. The pH was adjusted − − in vitro × + 3 Instead of studying isolated . glutamine, no pyruvate, supple- 13 1 NaCl–5 mmol L − Lof1 1 C. Sufficient 1 mol L µ C, using Dubelco’s modified Eagle’s ◦ − ◦ C. The pH of the sample was adjusted ◦ ,37 2 and the weight of the sample was adjusted 3 glucose, 0.58 g L 1 porcine pepsin added, after which the samples were − 1 − digestion C until use. Water or 150 mmol L ◦ Although single compound-induced changes in TEER give 80 insights in the cellular effects leadingthis to is the a changed highly physiology, reductionist approachWhole to food study the products effects are ofunrelated food. compounds a that combination can interact of andbioactive together various provoke effects, the which related can and be differentby from the the effects compounds induced in isolation. incubated for 30 min at 37 wileyonlinelibrary.com/jsfa porcine pancreatin and 0.5 mL of a mixture ofand sodium taurocholate sodium glycodeoxycholate (176 mmol L MATERIALS AND METHODS Cell culture ATCC Caco-2 cells were grown on membranes of 33.6 mm medium (DMEM; Gibco-Invitrogen,with 4.5 Bleiswijk, g L The Netherlands) added to raise pH to at least 5.8, followed by 0.95 mL of 4 g L to 30 g. Samples were centrifuged for 30 min at 3023 to 7.5 with NaHCO The supernatant was taken,− flushed with nitrogen and stored at ThinCert transwells, putCells in were seeded 24-well with 150 suspension culture plates. In vitro Jonagold applesRepresentative were wedges of obtained apple,50% from without of weight core a 150 were mmol L taken local and supermarket. (FBS; Hyclone PerBio, Etten-Leur, Thereached confluence Netherlands) within to 4 days 9.1%. of Cells seeding.passage Cells numbers were 30 used with to 54; mediumweek. was replaced three times per and mashed with aputina50 hand blender. mLtube,thepHwasadjustedto2withHCl,and0.667 Twenty gramsof of 40 sample g was L mL was used instead of apple for preparing Preparation of crude apple extract Representative wedges of Jonagoldtaken apples, and without 125% core, ofwas weight were added 150 mmol to L apple, obtain and mashed a with a similar hand blender. Samplesto dilution were transferred tubes as the and centrifuged for 30 min at 3023 with sodium hydroxide if necessary, using phenol red in DMEM Exposure Samples were diluted 1 : 1 with DMEM until use. sample was adjusted to pH 6.5 with NaHCO describe the effect ofTEER-enhancing crude effects apple on Caco-2 extract cells. on TEER and show its mented with heat-inactivated (45 min 56 compounds, we studied the effect of crudeSome food work extracts has on been TEER. donelike to study extracts the of effect ginger, of sweetshowed whole pepper, either products, no and effect, edible or burdock, a which decrease in TEER. supernatant was taken, flushed with nitrogen and stored at was flushed withincubated nitrogen for and 1 h the at sample 37 was subsequently for 21 days at 5% CO

440 441 5. = will be in vitro n 20 Assuming SD, ML-7 on its 19 ± We tested the 24,25 8 Indicates a statistical ∗ digested wedges of * 05. Averages Cortex Skin . we had expected to find 0 In vitro wileyonlinelibrary.com/jsfa < or at concentrations reported P 21,22 23 * wedge digested salt control. Representative digested apple parts on Caco-2 TEER expressed in vitro apple already resulted in an increase of TEER. This in vitro and Jonagold apples contain quercetin glycosides 1 8 − Control Effect of 0

80 60 40 20

200 180 160 140 120 100 TEER (% of control) of (% TEER We were interested to know whether the increase in epithelial An apple can be roughly divided into three morphological parts: significant difference from control with concentration represents a dilution factora of more three- than to 10, five-fold while dilution is expected in humans. in the skin and not in the cortex, Figure 2. as % relative to an fresh weight L needed to affirm the resistance altering effect of apple in humans. resistance persisted after removal ofapple-containing the apple sample extract. was When removed the returned after to control 4 levels h within 30 exposure, minthus (Fig. TEER 1c). the Apple extract ability has to inducewithout a an durable enhanced effect. TEER in Caco-2 cells, but the skin, the cortex (flesh) and the applethe core, of cortex which the are skin commonly and usedthe for effect consumption. To can test be whether cortex related tissue to were specific tested parts separately. apple, of the containing apple, cortex skinmonolayer, and and as skin, did increased appledigested TEER apple cortex skin, of when alone added a in (Fig.not equal Caco-2 2). result amount in to However, a the cells statistical did The significantly TEER-enhancing activity increase of of apples is the thus mainly TEER situatedcortex in (Fig. the and 2). to, a lesser extent,quercetin on and a quercetin weight glycosides basis, have in shownin Caco-2 the to cells, skin. enhance Since TEER a similar dose–response range inepithelium,itisnotunlikelythatasimilarresponsecanbetriggered Caco-2 cells as for humanin small the human intestinalhumans epithelium. using a However, dual experiments sugar or with Cr-EDTA uptake approach most TEER-enhancing activity in theargue skin. against These quercetin results therefore glycosidesThe being nature the of the active TEER-enhancing compound. ingredient is still unknown. Regulation of the apple-induced increase in TEER Induction of TEER has beenphosphokinase shown inhibitors to H7 be inhibited and by staurosporin. the broad to have an effect on TEER of Caco-2 monolayers. inhibiting activity of these inhibitors on apple-induced paracellular resistance together withan intracellular other calcium knowninhibitors chelator blocked kinase (Table 1). the inhibitorsconcentrations None apple-induced used and of increase (Fig. the 3). ofto tested The the TEER inhibitors cells at have atreported the dissociation concentrations been constants, that applied are much higher than their in vitro In vitro digested ) –1 2011 Society of Chemical Industry * * c in vitro * ** * digestion. The digested apple and TEER * in vitro in vitro digestion control ( * * * digested apple in sample (g L in vitro Time of exposure (h) Time of exposure (h) in vitro digested apple as could be observed after : 439–444 Indicates significant difference from control with ∗ medium-> apple apple-> medium 92 digestion control digested apple 3. digested apple. A straight line is fitted through the = in vitro Amount of n in vitro in vitro crude apple extract DMEM only 2012; in vitro SD, 0 50 100 150 200 250 ± -3 -2 -1 0 1 2 3 4 5 Modification of TEER by apple. (a) Change of TEER of Caco-2 -2-1012345 0 0 80 60 40 20 75 50 25 90 80

05. 160 140 120 100 200 175 150 125 100 140 130 120 110 100

.

TEER (% of of (% TEER = 0) = t 0 TEER (% of of (% TEER = 0) = t TEER (% of of (% TEER = 0) = t TEER enhancing activity of apple extract is linearly related to (c) (b) < (a) Apple induces higher epithelial 1 resistance www.soci.org data points. (c) Addition and removaladdition of apple and extract. Moments removal of sample Averages are indicated. Symbols are as given in panel (a). salt solution), crude applemeasured extract, over and time. (b)amounts Change of in TEER upon 2 h exposure to different P J Sci Food Agric the amount of digestion protocol included pH changes, enzymatic digestion with pepsin and pancreas enzymes,digested and apple addition showed of a bile comparable TEER-enhancingcrude salts. effect extract as (Fig. a 1), indicatingcan that pass the the TEER human gastric enhancing environment. activity exposures of the dilutions series (Fig. 1b). A concentration of 74 g apple extracts ranged from 20depending to on 90% the of seeding theshown). Since original batch food is resistance, subjected of to various thereaches digestive steps the Caco-2 before small it cells intestine, for (data whichas the not a Caco-2 model, cell layer we is testedapple used whether homogenate the remained induction after of TEER by a crude Figure 1. cells after exposure to DMEM, , . et al : 439–444 92 2012; ) Reference 1 − J Sci Food Agric mol L µ Time of exposure (h) Time of exposure (h) DMEM Apple GFX Apple + GFX DMEM Apple ST Apple +ST 3. = n -3 -2 -1 0 1 2 3 4 5 -3 -2 -1 0 1 2 3 4 5 0 0

80 60 40 20 80 60 40 20

160 140 120 100 160 140 120 100 SD,

= 0) = t of (% TEER = 0) = t TEER (% of of (% TEER ± in paracellular resistance therefore did not involveA, phosphokinase phosphokinase C, phosphokinase G, myosinor light intracellular calcium chain signalling. kinase, Effect of apple on tight-junction-related gene expression Paracellular transport isthe regulated cells. by In tight order junctions to between analyse the genes involved in the tight

(b) (d) Time of exposure (h) www.soci.org RAM Vreeburg 2011 Society of Chemical Industry Apple BAPTA-AM Apple + BAPTA-AM DMEM c -tetraacetic acid tetrakis(acetoxymethyl ester).  -3 -2 -1 0 1 2 3 4 5 N , 0  80 60 40 20 N , 160 140 120 100

N TEER (% of of (% TEER = 0) = , t N (e) -hexahydro-1,4-diazepine hydro-chloride. H Time of exposure (h) Time of exposure (h) DMEM Apple H7 Apple + H7 DMEM Apple d ML-7 Apple + ML-7 -3 -2 -1 0 1 2 3 4 5 -3-2-1012345 0 0

80 60 40 20 80 60 40 20

160 140 120 100 160 140 120 100

= 0) = t of (% TEER TEER (% of of (% TEER = 0) = t (c) (a) Phosphokinase inhibitors used for pharmacological study in TEER regulation Effectofphosphokinaseinhibitorsandanintracellularcalciumchelatoronapple-inducedTEER.(a) H7,(b) GF109203X,(c) ML-7,(d) staurosporine GF 1029230X, 3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride. BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane- H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine. ML-7, 1-(5-iodonaphthalene-1-sulfonyl)-1 H-7ML-7GF 109203XStaurosporineBAPTA-AM Phosphokinase A Broad and spectrum Broad C phosphokinase spectrum inhibitor inhibitor phosphokinase inhibitor Myosin light chain kinase inhibitor Intracellular calcium chelator 200 0.2 2 30 10 22 24 22 22 23 Table 1. Compound Type of inhibitor Conc. used ( wileyonlinelibrary.com/jsfa (e) BAPTA-AM. See Table 1 for abbreviations and concentrations used. Average own enhanced the TEERof of the apple Caco-2 extract monolayer,apple-induced but increase increased addition in TEER the wasML-7 TEER similar as for for even cells cells treated without(Fig. more with 3c). the (Fig. myosin The 3c). light chain apple-inducedstatistically An different kinase in TEER the inhibitor presence or enhancement absence ofcalcium the was intracellular chelator BAPTA-AM also (Fig. 3e). not An apple-induced increase Figure 3.

442 443 ∗ 9 1.13 0.88 1.09 1.13 1.17 1.27 0.82 1.27 1.37 1.38 0.93 0.91 0.90 1.16 0.56 0.94 Fold induction wileyonlinelibrary.com/jsfa 05 . 0 < digestion apple, showing a 2-fold induction ) 1.13 ) 1.15 digestion simulation, and is mainly localised in the in vitro ) 1.29 ) 0.92 expression could be a preparation for the incorporation C. elegans in vitro C. elegans C. elegans extract, and (Fig. 4). However, given theof fast apple reduction from in TEER theincorporation upon incubation of removal mixture claudin (Fig. 4 1c), proteinsare it in the the is tight mechanisms unlikely junction that by complex CLDN4 which the TEERof is claudin increased. 4 into Enhanced tightparacellular junctions resistance, as for proposed the by long-term Suzuki and adjustment Hara. of cortex. The localisationcortex of argues the forglycosides, TEER-enhancing an which are active factor present compound in in other the the than skin quercetin only. Apple-induced CONCLUSION Apple can induce an increase inas TEER paracellular in resistance Caco-2 measured cells. Thisafter TEER enhancing activity is maintained Drosophila ) 1.28 ) 0.84 ) 1.06 digestion controls, with a false discovery rate 2011 Society of Chemical Industry ) Drosophila c C. elegans C. elegans (formerly named in vitro in Caco-2 cells was expression has been Claudin 4 expression TJP2 Drosophila 9 CLDN4 and CLDN4 Rho GTPase activating protein 17 1.13 Claudin 1 Claudin 12 Rho/Rac guanine nucleotide exchange factor (GEF) 2Ash1 (absent, small, or homeotic)-like ( Claudin 16 1.10 Claudin 17 Claudin 2 Claudin 23 Claudin 4 Claudin 5 Claudin 7 Cingulin Coxsackie virus and adenovirus receptorHypothetical protein LOC283999 1.06 1.20 Crumbs homolog 3 ( InaD-like ( Shroom family member 2 Membrane associated guanylate kinase, WW and PDZMembrane domain associated containing guanylate 1 kinase, WW and PDZMICAL-like domain 2 containing 3 0.90 0.78 Immunoglobulin superfamily, member 5Lin-7 homolog A ( Multiple PDZ domain proteinMetadherin 0.88 1.16 Par-6 partitioning defective 6 homolog alpha ( Par-6 partitioning defective 6 homolog beta ( Par-6 partitioning defective 6 homolog gamma ( Protein kinase C, zeta Tight junction associated protein 1 (peripheral) 0.87 Lin-7 homolog C ( Membrane protein, palmitoylated 5 (MAGUK p55 subfamilyMembrane member protein, 5) palmitoylated 7 (MAGUK p55 subfamily member 7) 1.16 1.18 (zona occludens 1) (zona occludens 2)VAMP (vesicle-associated membrane protein)-associated protein A, 33 kDa 1.07 1.20 1.09 CLDN1 expressionwasinduced1.38-foldwhen : 439–444 digested apple and genes that are ascribed CLDN4 92 digested apple compared with 9 2012; in vitro in vitro Genes that are ascribed to the tight junction cellular compartment (GO:0005923) and whose expression in Caco-2 cells was different after Apple-induced expression of 26 ) was increased by apple exposure (Table 2). The expression A value of 1 represents no change in expression relative to a non-apple exposed control. CLDN1 CLDN12 ∗ CGN CLDN16 ASH1L ARHGAP17 ARHGEF2 Table 2. exposure to CLDN17 CLDN2 CLDN23 CLDN4 CLDN5 CLDN7 CRB3 F11R CXADR IGSF5 INADL LIN7A Gene symbol Gene name MAGI3 MICALL2 LOC283999 MAGI1 LIN7C MPDZ MPP7 MTDH MPP5 PARD6A PARD6B PARD6G PRKCZ SHROOM2 TJAP1 TJP2 VAPA TJP1 Apple induces higher epithelial 1 resistance www.soci.org validatedbyRT-qPCRinaseparateexperimentforbothcrudeapple J Sci Food Agric ZO-2 of these genes have been correlated witheffectofquercetin. the fast TEER enhancing junction a microarrayexposed experiment to wasto performed. the tight junction Cells cellular compartment,ontology were according to consortium the gene (GO:0005923),was were represented analysed. by This 67an genes group altered on expression in the response array, exposure togenes of is apple. given which The in list 37 Table of 2, 37 showed andclaudin includes several genes. of the Expression pore-forming of determined by microarray (Table 2); linked to long term TEER enhancement. in Madin–Darby canineexpression kidney of claudin (MDCK) 4 with II reducedsodium. paracellular cells permeability for have linked the , . J et al et al JNutr JNutr ,Effects Sci Hortic Eur J Nutr : 439–444 . et al :7211–7219 :1521–1536 92 J Roy Stat Soc :F1078–F1084 50 36 in vitro 285 :1982–1986 (2007). 2012; 30 :1096–1121 (1955). :1345–1346 (1994). 50 58 J Clin Invest J Agric Food Chem J Sci Food Agric :1860–1865 (2003). :124–129 (2011). Biol Pharm Bull 2 :3375–3386 (2000). 133 J Am Stat Assoc :35760–35765 (2002). 113 Am J Physiol – Renal Physiol 277 JNutr :1927–1932 (2010). :289–300 (1995). :154–158 (1999). Food Funct 58 57 43 Biosci Biotechnol Biochem JCellSci J Biol Chem , The nondigestible disaccharide epilactose increases :1557–1563 (2010). :965–974 (2009). :249–263 (2000). :183–191 (2008). 83 concentration of polyphenolic antioxidants inof apple existing juice. production 1. Effect methods. (2002). Myosin light chaindissemination. kinase plays an essentialmechanisms role involved in in docosahexaenoicin acid-induced S-flexneri increases tight junction140 permeability in caco-2 cell monolayers. Phospholipase C-gammapermeability modulates through epithelialproteins. hyperphosphorylation tight of junction tight junction acid levels in apple fruit: characterisation of variation. selectivity in cation or anion-selective epithelialof lines different by claudins. expression Series B-Method (1957). et al paracellular Ca absorption vialight Rho-associated chain kinase- kinase-dependent and mechanisms myosin inAgric rat Food small Chem intestines. practical and powerful approach to multiple testing. digestion and absorption in the human. 139 Tol EAF, Polyunsaturatedintegrity fatty and reduce IL-4 acids mediated permeability support epithelial barrier (2003). through theclaudin-1 assembly and of the expression zonnula of occludens-2, claudin-4 in , caco-2 and cells. of capsaicin onhuman cellular intestinal Caco-2 damage cells. and monolayer permeability in Apple pectin and aeffective polyphenol-rich together apple than concentrateplasma lipids are separately in more rats. on cecal fermentations and based backgroundarrays. adjustment Working Papers for 2004. Dept.University, oligonucleotide p. of 1 Biostatistics, (2004). expression Johns Hopkins treatments with a control. 47 Nahrung (Food) on the transepithelial permeability ofcell the monolayer. human intestinal Caco-2 quantitative RT-PCR inexposure differentiated studies. Caco-2 cells used for food 9 Suzuki T and Hara H, Quercetin enhances intestinal barrier function 22 van der Sluis AA, Dekker M, Skrede G and Jongen WMF, Activity and 23 Rathman M, de Lanerolle P, Ohayon H, Gounon P24 and Sansonetti Roig-Perez P, S, Cortadellas N, Moreto M and Ferrer R,25 Intracellular Ward PD, Klein RR, Troutman MD, Desai S and Thakker DR, 21 Awad MA, de Jager A and van Westing LM, Flavonoid and chlorogenic 26 Van Itallie CM, Fanning AS and Anderson JM, Reversal of charge 20 Suzuki T, Nishimukai M, Takechi M, Taguchi H, Hamada S, Yokota A, 18 Benjamini Y and Hochberg Y, Controlling the false discovery rate –19 a Borgstrom B, Dahlqvist A, Lundh G and Sjovall J, Studies of intestinal 10 Willemsen LEM, Koetsier MA, Balvers M, Beermann C, Stahl B and van 12 Tsukura Y, Mori M, Hirotani Y, Ikeda K, Amano F, Kato R, 16 Wu Z, Irizarry RA, Gentleman R, Murillo FM and Spencer F, A model 17 Dunnett CW, A multiple comparison procedure for comparing several 13 Aprikian O, Duclos V, Guyot S, Besson C, Manach C, Bernalier A, 11 Shimizu M, Modulation of intestinal functions by food substances. 14 Hashimoto K,Matsunaga NandShimizu M,Effectofvegetableextracts 15 Vreeburg RAM, Bastiaan-Net S and Mes JJ, Normalization genes for 3. = n Clinical www.soci.org RAM Vreeburg SD, 05. . 2011 Society of Chemical Industry ± 0 c < * P :1067–1073 (2008). Drug Discovery Today expression. Expression 138 J Allergy Clin Immunol expression. These results JNutr CLDN4 :241–256 (2007). 23 :465–478 (2007). CLDN4 :403–429 (2006). 38 Ann Rev Pathol – Mechanisms Disease 68 models of intestinal epithelial cell , New diseases derived or associated with the * Cell Biol Toxicol et al In vitro Arch Med Res :20–28 (2011). 41 Ann Rev Physiol Crude apple extract In vitro digested apple , Quercetin enhances epithelial barrier function and increases :3–20 (2009). Effect of 4 h apple exposure on :395–408 (2005). :119–144 (2010).

transport. 124 differentiation. regulation and disease pathogenesis. Kedinger M, et al claudin-4 expression in Caco-2 cells. Exp Allergy homeostasis and disease. 5 and endothelial barriers in human disease. 10 Laffe I, Ruiz A, tight junction.

3 2 1 0 Relative CLDN4 induction CLDN4 Relative 2 Van Itallie CM and Anderson JM, Claudins and epithelial paracellular 6 Groschwitz KR and Hogan SP, Intestinal barrier function: Molecular 1 Simon-Assman P, Turck N, Sidhoum-Jenny M, Gradwohl G and 8 Amasheh M, Schlichter S, Amasheh S, Mankertz J, Zeitz M, Fromm M, 3 Marchiando AM, Graham WV and Turner JR,4 Epithelial Mullin barriers JM, Agostino in N, Rendon-Huerta E and Thornton JJ, Epithelial 7 Perrier C and Corthesy B, Gut permeability and food allergies. 5 Cereijido M, Contreras RG, Flores-Benitez D, Flores-Maldonado C, Indicates significant difference from the control, with wileyonlinelibrary.com/jsfa REFERENCES ACKNOWLEDGEMENTS We thank Mechteld Grootte Bromhaar, Mark Boekschoten, andMadMaxmicroarrayanalysisteamfortheirhelpwiththemicroarray the experiments. This workUniversity and Research was Centre IPOP systems supported biology program. by the Wageningen TEER enhancement is not inhibitedand by is phosphokinase correlated inhibitors with increase Figure 4. ∗ indicate that foodintestine. might Human modulate trials the willand be barrier needed establish function to a of confirm scientificaltered these the base barrier findings function for is dietary desired. advice in cases where values are corrected for expression of controls. Averages

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