Confluent Monolayers Of Caco-2 Cells Research Articles

Inhibition of Transglutaminase 2 as a Therapeutic Strategy in Celiac Disease-In Vitro Studies in Intestinal Cells and Duodenal Biopsies.

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 µM, 15 ± 3 vs. 48 ± 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 µM; 12 ± 7% vs. 45 ± 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 µM, 25 ± 13% vs. 22 ± 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 µM. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD.

Cryptosporidium parvum infection induces autophagy in intestinal epithelial cells.

Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP-induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin-4 and E-cadherin. Therefore, we utilised confluent Caco-2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco-2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 106 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co-treated with CP + CQ as compared to CP or CQ alone, suggesting that CP-induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin-4 and E-cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP-induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis.

An intestinal paracellular pathway biased toward positively-charged macromolecules

Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). While TNF-α/IFN-γ treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes.

Effect of adenovirus mediated human intestinal trefoil factor gene on intestinal epithelial cells

Objective To construct a recombinant adenovirus vector of human intestinal trefoil factor(hITF)and observe its effect on human intestinal epithelial cells(Caco-2). Methods A recombinant adenovirus vector containing hITF gene was constructed and infected Caco- 2 cells. The expression of hITF mRNA and protein were detected by reverse transcriotion-polymerase chain reaction(RT-PCR), Western blotting and immunofluorescence. Then, wounds were established in confluent monolayers of Caco-2 cells infected with recombinant adenovirus vector, and the width of the cells migration was measured in a blind manner. Results The recombinant adenovirus vector Ad-hITF was constructed and the infection efficiency was about 100%. RT- PCR and Western blotting assay demonstrated that hITF was transcribed and expressed successfully. hITF was proved to distribute in cytoplasm by immunofluorescence assay. In addition, Ad-hITF enhanced migration activity of HT-29 cells obviously. Conclusion Recombinant adenovirus vector containing hITF gene was successfully constructed and its effect was confirmed in promoting cells migration. This research lay foundations for the further application of hITF gene therapy. Key words: Intestinal trefoil factor; Adenovirus; Intestinal epithelial cells; Migration

Interleukin 17A evoked mucosal damage is attenuated by cannabidiol and anandamide in a human colonic explant model

Interleukin 17A (IL-17A) is a cytokine linked to inflammatory bowel disease. We investigated IL-17A expression in human colonic mucosa, whether IL-17A can elicit colonic mucosal damage in a human explant model and modulate gastrointestinal epithelial permeability in cell culture. We also tested if select cannabinoid ligands, shown to be protective in colitis models could attenuate damage caused by IL-17A. In addition, the ability of pro-inflammatory cytokines TNF-α and IL-1β to modulate levels of IL-17A in the explant colitis model was also explored. IL-17A incubation caused significant mucosal epithelial and crypt damage which were attenuated following hydrocortisone treatment, and also reduced following anandamide or cannabidiol incubation. IL-17A-evoked mucosal damage was also associated with an increase in matrix metalloprotease activity. However, IL-17A did not induce any significant changes in epithelial permeability in confluent Caco-2 cell monolayers over a 48h incubation period. IL-17A was located predominantly in human mucosal epithelium together with IL-17C, but both IL-17A and IL-17C were also expressed in the lamina propria and submucosa. Incubation of human colonic mucosal tissue or Caco-2 cells with pro-inflammatory cytokines TNF-α and IL-1β however did not alter IL-17A expression. These results indicate IL-17A has a widespread distribution in the human colon and the capacity to elicit mucosal damage which can be attenuated by cannabinoid ligands.

Multidrug resistant proteins mediate extracellular ATP release in Caco2 cell monolayers

Epithelial ATP release is an essential component of extracellular purinergic secretory regulation; ATP transporting channels, pannexin (Panx) hemichannels and multidrug resistant proteins (MDR/MRP) have been implicated. The robust intestinal ecto‐ATPase activity present in Caco‐2 cells impairs measurements of medium ATP. We thus transfected c‐Myc‐tagged GPI‐anchored plasma membrane luciferase (pmeLuc) to Caco2 cell monolayer to monitor ATP release at the cell surface. Confluent Caco‐2 cell monolayers were transfected with pmeLuc, confirmed by immunolocalization. Although transfection efficacy into Caco2 cell is ~ 10 %, much less than in HEK293 ~ 80 %, luminescence was measurable over the background (~ 3000 in Caco‐2, ~ 30,000 in HEK293). Luminescence was stable over 40 min, suggesting constitutive ATP release. Among the pharmacological reagents reported as inhibitors for ATP release, MK571, a specific MDR/MRP inhibitor dose‐dependently inhibited ATP release. Stilbene anion transport inhibitors or Panx inhibitors had no effect. Vesicles expressing P‐glycoprotein (Mdr1) and MRP4, but not MRP2 accumulated ATP inside. MDR/MRP mediates ATP release in intestinal epithelia, a novel mechanism for transmembrane ATP transport. Regulation of ATP release may have important implications for the physiological functioning of intestinal and other secretory epithelia. NIH‐NIDDK R01 DK54221 &amp; R21 DK081199

Cannabinoids mediate opposing effects on inflammation‐induced intestinal permeability

Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro. Confluent Caco-2 cell monolayers were treated for 24 h with IFNγ and TNFα (10 ng·mL(-1) ). Monolayer permeability was measured using transepithelial electrical resistance and flux measurements. Cannabinoids were applied either apically or basolaterally after inflammation was established. Potential mechanisms of action were investigated using antagonists for CB(1) , CB(2) , TRPV1, PPARγ and PPARα. A role for the endocannabinoid system was established using inhibitors of the synthesis and degradation of endocannabinoids. Δ(9) -Tetrahydrocannabinol (THC) and cannabidiol accelerated the recovery from cytokine-induced increased permeability; an effect sensitive to CB(1) receptor antagonism. Anandamide and 2-arachidonylglycerol further increased permeability in the presence of cytokines; this effect was also sensitive to CB(1) antagonism. No role for the CB(2) receptor was identified in these studies. Co-application of THC, cannabidiol or a CB(1) antagonist with the cytokines ameliorated their effect on permeability. Inhibiting the breakdown of endocannabinoids worsened, whereas inhibiting the synthesis of endocannabinoids attenuated, the increased permeability associated with inflammation. These findings suggest that locally produced endocannabinoids, acting via CB(1) receptors play a role in mediating changes in permeability with inflammation, and that phytocannabinoids have therapeutic potential for reversing the disordered intestinal permeability associated with inflammation. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ∼90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.

M1850 Geranylgeranylacetone Prevents Cytokine-Induced Down-Regulation of Heat Shock Protein 70 and Ameliorates Oxidative Injury of Colonic Epithelial Cells In Vitro

Background and Aims: Heat shock protein (HSP) 70 plays an important role in protection of gastrointestinal cells from a variety of stresses. Recent studies have shown that HSP70 is upregulated in inflamed intestinal mucosa in patients with inflammatory bowel diseases(IBD)(Luding, Dig Dis Sci, 1999), while other studies have shown that proinflammatory cytokines, which are highly expressed in intestinal mucosa in IBD patients, down-regulate expression of HSP 70(Sien H, Gastroenterology 2007). In the present study, we examined if proinflammatory cytokines inhibit HSP 70 expression in colonic mucosa, thereby exaggerating oxidative injury in colonic mucosa. We also examined if induction of heat shock protein 70 (HSP 70) by geranylgeranyacetone (GGA), a non-toxic HSP inducer, could protect colonic mucosa from oxidative injury In Vitro.Methods: Confluent monolayers of Caco-2 cells were incubated on membrane permeable supports. HSP 70 expression was evaluated by Western blot analysis and real time PCR. Cell viability was assessed by MTT assay. Apoptosis was evalutated by Caspase-3 assay. Integrity of the Caco-2 cell monolayers was assessed by measuring electrical transepithelial electrical resistance (TER) and transmucosal flux of FITClabeled inulin across the monolayers. Cells were incubated with or without cytokines (IFNγ and TNF-α) and/or Hydrogen peroxide (H2O2). In some experiments, the cells were preincubated with GGA. Results: 1. Treatment with either IFN-γ alone or TNF-α alone did not affect either HSP 70 expression or cell viability, and did not affect TER or FITC-labeled inulin flux in the Caco-2 cell monolayers. 2. Combination of IFN-γ (100 nM) and TNF-α (100 nM) downregulated HSP 70 expression, but did not affect cell viability or mucosal integrity. 3. In the absence of the cytokines, lower doses of H2O2 (< 10 mM) upregulated HSP 70 expression, and did not cause cell death or disruption of the mucosal integrity. 4. In the presence of the cytokine cocktail, lower doses of H2O2 failed to increase in HSP 70 expressions, enhanced cell death, and impaired mucosal integrity. 4. Pretreatment of the cells with GGA up-regulated HSP 70 expression, and prevented oxidative injury induced by H2O2, in both the absence and the presence of the cytokine cocktails. Conclusions: The present study suggest that (1)IFN-γ and TNF-α synergistically downregulate HSP 70 expression in colonic mucosa, and exaggerate colonic mucosal injury induced by oxidative stress, (2)induction of HSP70 by GGA appears to be useful in treatment and/or prevention of colonic mucosal injury in IBD patients.

M1851 Roles of Cyclooxygenase/Prostaglandin E2, Nitric Oxide Synthase/Nitric Oxide, and Enterobacteria in Pathogenesis of Ischemic Enteritis in Rats

Background and Aims: Heat shock protein (HSP) 70 plays an important role in protection of gastrointestinal cells from a variety of stresses. Recent studies have shown that HSP70 is upregulated in inflamed intestinal mucosa in patients with inflammatory bowel diseases(IBD)(Luding, Dig Dis Sci, 1999), while other studies have shown that proinflammatory cytokines, which are highly expressed in intestinal mucosa in IBD patients, down-regulate expression of HSP 70(Sien H, Gastroenterology 2007). In the present study, we examined if proinflammatory cytokines inhibit HSP 70 expression in colonic mucosa, thereby exaggerating oxidative injury in colonic mucosa. We also examined if induction of heat shock protein 70 (HSP 70) by geranylgeranyacetone (GGA), a non-toxic HSP inducer, could protect colonic mucosa from oxidative injury In Vitro.Methods: Confluent monolayers of Caco-2 cells were incubated on membrane permeable supports. HSP 70 expression was evaluated by Western blot analysis and real time PCR. Cell viability was assessed by MTT assay. Apoptosis was evalutated by Caspase-3 assay. Integrity of the Caco-2 cell monolayers was assessed by measuring electrical transepithelial electrical resistance (TER) and transmucosal flux of FITClabeled inulin across the monolayers. Cells were incubated with or without cytokines (IFNγ and TNF-α) and/or Hydrogen peroxide (H2O2). In some experiments, the cells were preincubated with GGA. Results: 1. Treatment with either IFN-γ alone or TNF-α alone did not affect either HSP 70 expression or cell viability, and did not affect TER or FITC-labeled inulin flux in the Caco-2 cell monolayers. 2. Combination of IFN-γ (100 nM) and TNF-α (100 nM) downregulated HSP 70 expression, but did not affect cell viability or mucosal integrity. 3. In the absence of the cytokines, lower doses of H2O2 (< 10 mM) upregulated HSP 70 expression, and did not cause cell death or disruption of the mucosal integrity. 4. In the presence of the cytokine cocktail, lower doses of H2O2 failed to increase in HSP 70 expressions, enhanced cell death, and impaired mucosal integrity. 4. Pretreatment of the cells with GGA up-regulated HSP 70 expression, and prevented oxidative injury induced by H2O2, in both the absence and the presence of the cytokine cocktails. Conclusions: The present study suggest that (1)IFN-γ and TNF-α synergistically downregulate HSP 70 expression in colonic mucosa, and exaggerate colonic mucosal injury induced by oxidative stress, (2)induction of HSP70 by GGA appears to be useful in treatment and/or prevention of colonic mucosal injury in IBD patients.

THE SYNERGISTIC EFFECT OF ETHANOL AND SHOCK INSULTS ON CACO2 CYTOKINE PRODUCTION AND APOPTOSIS

Gut epithelial cells are important in orchestrating immunoinflammatory responses in the gut and may impact systemic immunocompetent cells after shock and trauma. Ethanol (EtOH) intoxication is an important etiological factor in trauma and may increase the likelihood of posttraumatic septic complications. Both EtOH and gut I/R impair intestinal barrier function. However, their combined effects on intestinal epithelial cell function and barrier integrity are unknown. Confluent CaCO2 cell monolayers were grown in a two-chamber culture system and exposed to 0.1% EtOH and/or Escherichia coli C-25 under normoxic (21% O2) or hypoxia (5% O2) followed by reoxygenation (H/R). Apical and basal compartment supernatants were collected, and TNF and IL-6 were quantitated by enzyme-linked immunosorbent assay (picograms per milliliter). CaCO2 cell integrity was indexed by apoptosis and monolayer permeability. TNF-alpha production by CaCO2 cells are greatest when incubated with EtOH and then exposed to H/R group. The apical levels of TNF production are consistently higher than basal levels, although the trend toward increased cytokine production is similar in both compartments. IL-6 production by the CaCO2 cell is also greatest when CaCO2 cells incubated with EtOH undergoes H/R. Lastly, the findings in apoptosis mirror the data of the TNF production in the apical compartment. Ethanol and H/R have a synergistic effect on cytokine production and barrier dysfunction in this model. They may also contribute to increased infectious complications and posttraumatic organ failure.

Changes in macromolecular transport appear early in Caco-2 cells infected with a human rotavirus

Objective. Rotavirus is a major cause of viral gastroenteritis, but its interaction with intestinal mucosa is poorly understood. The aim of this study was to examine the effect of Wa rotavirus (VP7 serotype 1) on barrier function in confluent Caco-2 cell monolayers. Wa is the most common serotype causing severe diarrhoea in humans. Material and methods. We examined light and electron microscopic morphology, macromolecular transport, paracellular permeability, electrical parameters, disaccharidases and cytoskeletal structure in Wa- and in control sham-infected cells using a homologous human virus-cell system resembling human infection. Results. During the first 48 h following Wa infection, there was no evidence of loss of integrity or of cytopathic effect in the monolayer. A significant cytopathic effect was noticed after 48 h. Further studies examined the initial 24-h period during which there was no evidence of significant injury. Apical-to-basolateral transcytosis of the macromolecule horseradish peroxidase (HRP) was selectively inhibited at 4 and 24 h post-infection with Wa. There were no significant changes in basolateral-to-apical transcytosis, endocytosis or in apical-to-apical recycling of HRP after Wa infection. G- and F-actin levels were significantly reduced within an area corresponding to the viroplasm in Wa-infected cells but not elsewhere in the monolayer. Conclusions. The early stages of rotavirus infection, before gross epithelial injury, are associated with a selective reduction in the apical uptake and transcytosis of macromolecules. We speculate that this is an epithelial defence mechanism.

Role of phosphatidylcholine saturation in preventing bile salt toxicity to gastrointestinal epithelia and membranes

The mechanism which protects the biliary and intestinal mucosa from the detergent properties of bile acids is not fully understood. We employed three contrasting in vitro model systems (human red blood cells, polarized intestinal [Caco-2] cells, and synthetic liposomes), to compare the efficacy of saturated and unsaturated phosphatidylcholine (PC) to protect cells and membranes from bile salt injury. Hemolysis of red blood cells, electrical resistance across confluent monolayers of Caco-2 cells, and disruption of synthetic PC liposomes were assessed after incubation with varying concentrations of bile salt (sodium deoxycholate) alone or in the presence of saturated or unsaturated PC. The hemolytic activity of deoxycholate on red blood cells was observed at > or =2 mM, and could be blocked by equimolar concentration or greater of both saturated or unsaturated PC. In contrast, exposure of Caco-2 cells to deoxycholate at > or =0.8 mM induced a maximal decrease in resistance, which was reversed by > or =0.8 mM unsaturated PC or 5 mM saturated PC. Similarly, synthetic liposomes were permeabilized by 0.8 mM deoxycholate and were protected by a lower concentration of unsaturated PC (2 mM) than saturated (5 mM). Cells can show variable resistance to bile salt toxicity. Extracellular PC, especially in the unsaturated state, can directly protect cell and artificial membranes from bile salt injury. These findings support a role for biliary PC in the formation of mixed micelles that have low cytotoxic properties.

Danofloxacin-mesylate is a substrate for ATP-dependent efflux transporters.

Next to its broad antimicrobial spectrum, the therapeutic advantages of the fluoroquinolone antimicrobial drug Danofloxacin-Mesylate (DM) are attributed to its rapid distribution to the major target tissues such as lungs, intestines and the mammary gland in animals. Previous analyses revealed that effective drug concentrations are achieved also in luminal compartments of these organs, suggesting that active transport proteins facilitate excretion into the luminal space. Members of the ATP-Binding Cassette (ABC) superfamily, including P-gp, BCRP and MRP2 are known to be expressed in many tissue barriers and in cell-membranes facing luminal compartments. Hence we hypothesized that DM is a substrate for one of these efflux-transporters. Confluent monolayers of Caco-2 cells, grown on microporous membranes in two-chamber devices were used. DM concentrations were measured by fluorimetric assay after HPLC of the culture media. DM transport across Caco-2 cells was asymmetric, with a rate of secretion exceeding that of absorption. The P-gp inhibitors PSC833 and GF120918 and the MRP-inhibitor MK571 partially decreased the secretion of DM and increased its absorption rate. The BCRP inhibitor, Ko143, decreased secretion only at a concentration of 1 microM. When DM was applied together with ciprofloxacin, secretion as well as absorption of DM decreased. DM is a substrate for the efflux transporters P-gp and MRP2, whereas the specific role of BCRP in DM transport needs further evaluation. These findings provide a mechanistic basis for the understanding of the pharmacokinetics of DM in healthy and diseased individuals.

Transport, deglycosylation, and metabolism of trans-piceid by small intestinal epithelial cells

Numerous epidemiological and animal studies have shown that consumption of red wine is related to reduced incidence of cardiovascular diseases and cancer. Trans-resveratrol (3, 5, 4'-trihydroxystilbene), a phenolic compound present in wine, has been reported to have a potential cancer chemopreventive activity. Moreover, it may exert a protective effect against atherogenesis through its antioxidant properties. Trans-piceid (3-ss glucoside of trans-resveratrol) is present to a greater extent than its aglycone in red wine, but hydrolysis of this glycosylated derivative can occur in small intestine and liver, which would enhance the amount of the biological active trans-resveratrol. The present study aimed to investigate the rate of transepithelial transport of trans-piceid using human intestinal Caco-2 cell monolayers and metabolism of this compound during its absorption across the small intestine. The transport of trans-piceid was evaluated in the human epithelial cell line Caco-2, which possesses enterocyte-like properties in vitro. For transepithelial experiments, confluent monolayers of Caco-2 cells were grown on Transwell inserts. For metabolic studies, we used both Caco-2 cells seeded on 6-well plates and rat small intestine cell-free extracts. The time course of apical (AP) to basolateral (BL) transport of trans-piceid showed that the favorable apparent permeability coefficient (Papp) declined rapidly during the 6 h of the experiment. This observation could be correlated with the appearance of metabolites. After incubation of Caco-2 cells with trans-piceid, trans-resveratrol was detected on both AP and BL sides. By using protein extracts obtained from rat, we conclude that the Lactase Phlorizin Hydrolase (LPH) and Cytosolic-ss-Glucosidase (CBG) are involved in the hydrolysis of trans-piceid. Furthermore, we show that after deglycosylation, the resulting aglycone is metabolized in trans-resveratrol-3-O-ss-glucuronide and to a lesser extent in trans-resveratrol-4'-O-ss-glucuronide, and that UGT1A1 is mainly involved in this metabolism. This study demonstrates that the transepithelial transport of trans-piceid occurs at a high rate and that the compound is deglycosylated in trans-resveratrol. There are two possible pathways by which trans-piceid is hydrolyzed in the intestine. The first is a cleavage by the CBG, after passing the brush-border membrane by SGLT1. The second is deglycosylation on the luminal side of the epithelium by the membrane-bound enzyme LPH, followed by passive diffusion of the released aglycone, which is further metabolized inside the cells into two glucuronoconjugates.

Identification of Antiadhesive Fraction(s) in Nonimmunized Egg Yolk Powder: In Vitro Study

The presence of antiadhesive component(s) in the hen egg yolk against foodborne pathogens was anticipated from results of a previous animal study conducted by the authors. The previous work showed egg yolk powder without specific antibodies is effective in controlling Salmonella enteritidis,Salmonella typhimurium, and Escherichia coli O157:H7 colonization in laying hens. Therefore, this study was necessary to locate the activity and identify the effective component(s). In vitro experiments were conducted using confluent Caco-2 cell monolayers. S. enteritidis, S. typhimurium, and E. coli O157:H7 were investigated against the various extracted granule and plasma fractions in three different assays: adhesion elimination, adhesion prevention, and antimicrobial. This study revealed original findings and identified the protective yolk fraction against the foodborne pathogens as the granule component, high-density lipoproteins (HDL). The protective activity conveyed by HDL was confirmed to remain intact despite peptic and tryptic enzymatic digestion and to have antiadhesive but not antimicrobial effect.

Translocation of Clostridium difficile Toxin B across Polarized Caco‐2 Cell Monolayers Is Enhanced by Toxin A

Clostridium difficile is the etiological agent of antibiotic-associated diarrhea; the most common form of nosocomial infectious diarrhea. The basis for the shock-like systemic symptoms observed in severe cases of this infection are not known. It is hypothesized that the invasion of C difficile toxins A and/or B from the gut mucosa may contribute to these symptoms.A polarized tissue culture model employing Caco-2 cells grown on transwell inserts was established to study the translocation of purified C difficile toxins A and B. C difficile toxins were (125)I labelled and inoculated onto confluent polarized Caco-2 cell monolayers to study translocation dynamics. Electrical resistance measurements were used to monitor monolayer confluence and tight junction integrity. Samples were taken from the apical and basal sides of the insert, as well as the insert itself, and tested using the human foreskin fibroblasts cell cytotoxicity assay to monitor partitioning of the radiolabelled toxins. Toxin A produced a 50% reduction in electrical resistance in 3 h whereas the same concentration of toxin B required at least 7 h to achieve the same effect. Both toxins A and B were able to translocate across confluent monolayers of Caco-2 cells. The combination of toxin A and B together was synergistic with respect to promoting the translocation of toxin B. Although the addition of toxin A resulted in a 100% increase in the amount of toxin B able to translocate, no increases in toxin A translocation were observed. These findings suggest a model of pathogenesis in which C difficile toxin A facilitates the translocation of toxin B from the gut into submucosal areas where it may play a role in inflammatory damage.

Effect of iron treatment on nickel absorption and gene expression of the divalent metal transporter (DMT1) by human intestinal Caco-2 cells.

Divalent Metal Transporter 1 (DMT1) is a transmembrane transporter located at the apical membrane of enterocytes and implicated in the duodenal uptake of iron. Results from expression experiments in Xenopus oocytes indicate that DMT1 can mediate transport of a wide range of divalent metals other than iron. The aim of the present study was to examine the effect of iron treatment on the uptake and transepithelial movement of 63Ni and to correlate that to DMT1 messenger RNA (mRNA) levels in human intestinal Caco-2 cells. Twenty-one days after confluent Caco-2 cell monolayers were treated for 1 or 3 days with medium supplemented with Fe(NTA)2 or control medium and 63Ni transport and DMT1 gene expression were measured at both time points. Functional effects of the iron treatment were assessed by examining uptake and transepithelial movement of 59Fe. Iron treatment resulted in decreased DMT1 gene expression which correlated well with the uptake of 59Fe and 63Ni into fully differentiated Caco-2 cells. This indicates that DMT1 is responsible for the apical transport of these metals in the intestinal epithelium and suggests that adequate iron intake and status will limit nickel absorption.

Modulation of digoxin transport across Caco-2 cell monolayers by citrus fruit juices: lime, lemon, grapefruit, and pummelo.

To evaluate the effects of fresh lime, lemon, grapefruit, and pummelo juices on the transport of digoxin, a P-glycoprotein (P-gp) substrate, in Caco-2 cell monolayers. Bidirectional [3H]-digoxin fluxes across confluent Caco-2 cell monolayers were determined in 0-50% fruit juices at pH 7.4. Verapamil HCl (100 microM) served as positive control. Juice toxicity was evaluated by the 3-(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Apical-to-basal (A-to-B) digoxin flux was enhanced by 50% fruit juice in the order of lemon > lime > pummelo > grapefruit. The four fruit juices could be divided into two groups based on their effects on transepithelial electrical resistance (TEER), viability, and digoxin transport activity of the Caco-2 cells. Grapefruit and pummelo juices produced similar digoxin transport profiles that were characteristic of those observed with P-gp inhibitors. Both juices decreased net digoxin efflux by 1.2 U per 10% increase in juice concentration and had a propensity to increase cellular TEER at high concentrations (>30%). However, cellular TEER and viability decreased with increasing concentration of lime and lemon juices. Both juices also produced similar digoxin transport profiles, the A-to-B and B-to-A digoxin Papp increasing with increasing juice concentration above 5%. Net digoxin efflux was 30% of control value and relatively independent of juice concentration. These results paralleled the groupings of the four fruits according to their prominent flavonoid pattern and taxonomy. The effects of lime, lemon, grapefruit, and pummelo juices on the TEER, viability, and digoxin transport activity of the Caco-2 cells appeared to be dependent on the dominant flavonoid pattern and taxonomy of the citrus fruits.

Effects of citicholine and dimethylsulfoxide on transepithelial transport of passively diffused drugs in the Caco-2 cell culture model

The objective of this study was to determine, using a Caco-2 cell monolayer model, the extent to which the paracellular and transcellular routes are altered by citicholine (CDP-Ch) and DMSO in the presence of human serum albumin (HSA). The apparent permeability (Papp) of mannitol in the presence of 4% (w/v) HSA was investigated using 0, 0.5, 1.0, 2.5, 5.0, and 10.0% (v/v)) of DMSO. The Papp for mannitol ranged from 0.56×10 −6 to 0.89×10 −6 cm/s (mean 0.77×10 −6). Increasing the concentration of DMSO does not appear to have an effect on the paracellular transport of mannitol and on the transepithelial resistance (TEER) of the monolayer, ( P>0.05). The effect of citicholine (CDP-Ch) was investigated in confluent Caco-2 cell monolayers incubated in the presence of 2, 4, 10, 40, 60, 100 and 200 mM CDP-Ch at 37 °C in an atmosphere of 7% CO 2 and 95% relative humidity. Papp of mannitol and diltiazem in the presence of CDP-Ch ranged from 0.53×10 −6 to 8.52×10 −6 cm/s and from 1.30×10 −5 to 2.71×10 −5 cm/s, respectively. CDP-Ch may have an effect on the stability of the tight junction complex resulting in an increase in the apparent permeability of mannitol.