Differentiated Caco-2 Monolayers Research Articles

Engagement of α3β1 and α2β1 integrins by hypervirulent Streptococcus agalactiae in invasion of polarized enterocytes.

The gut represents an important site of colonization of the commensal bacterium Streptococcus agalactiae (group B Streptococcus or GBS), which can also behave as a deadly pathogen in neonates and adults. Invasion of the intestinal epithelial barrier is likely a crucial step in the pathogenesis of neonatal infections caused by GBS belonging to clonal complex 17 (CC17). We have previously shown that the prototypical CC17 BM110 strain invades polarized enterocyte-like cells through their lateral surfaces using an endocytic pathway. By analyzing the cellular distribution of putative GBS receptors in human enterocyte-like Caco-2 cells, we find here that the alpha 3 (α3) and alpha 2 (α2) integrin subunits are selectively expressed on lateral enterocyte surfaces at equatorial and parabasal levels along the vertical axis of polarized cells, in an area corresponding to GBS entry sites. The α3β1 and α2β1 integrins were not readily accessible in fully differentiated Caco-2 monolayers but could be exposed to specific antibodies after weakening of intercellular junctions in calcium-free media. Under these conditions, anti-α3β1 and anti-α2β1 antibodies significantly reduced GBS adhesion to and invasion of enterocytes. After endocytosis, α3β1 and α2β1 integrins localized to areas of actin remodeling around GBS containing vacuoles. Taken together, these data indicate that GBS can invade enterocytes by binding to α3β1 and α2β1 integrins on the lateral membrane of polarized enterocytes, resulting in cytoskeletal remodeling and bacterial internalization. Blocking integrins might represent a viable strategy to prevent GBS invasion of gut epithelial tissues.

Cytoskeletal Responses and Aif-1 Expression in Caco-2 Monolayers Exposed to Phorbol-12-Myristate-13-Acetate and Carnosine.

The dis(re)organization of the cytoskeletal actin in enterocytes mediates epithelial barrier dys(re)function, playing a key role in modulating epithelial monolayer's integrity and remodeling under transition from physiological to pathological states. Here, by fluorescence-based morphological and morphometric analyses, we detected differential responses of cytoskeletal actin in intestinal epithelial Caco-2 cell monolayers at two different stages of their spontaneous differentiation, i.e., undifferentiated cells at 7 days post-seeding (dps) and differentiated enterocyte-like cells at 21 dps, upon challenge in vitro with the inflammation-mimicking stimulus of phorbol-12-myristate-13-acetate (PMA). In addition, specific responses were found in the presence of the natural dipeptide carnosine detecting its potential counteraction against PMA-induced cytoskeletal alterations and remodeling in differentiated Caco-2 monolayers. In such an experimental context, by both immunocytochemistry and Western blot assays in Caco-2 monolayers, we identified the expression of the allograft inflammatory factor 1 (AIF-1) as protein functionally related to both inflammatory and cytoskeletal pathways. In 21 dps monolayers, particularly, we detected variations of its intracellular localization associated with the inflammatory stimulus and its mRNA/protein increase associated with the differentiated 21 dps enterocyte-like monolayer compared to the undifferentiated cells.

Virulence traits and differential translocation of gut-derived Candida albicans

Candida albicansis an opportunistic pathogenic yeast commonly found as part of the gut microbiome, which is thought to be the major reservoir of C. albicans in humans. The ability of C. albicans to adhere to, invade and damage epithelium, along with subsequent escape from the gut lumen into the bloodstream, can lead to life-threatening disseminated fungal infections. Examining the interactions of C. albicans with the intestinal epithelial barrier is vital to understanding its ability to disseminate and cause systemic infections. A set of seven C. albicans strains (six of gut origin: one healthy donor, one cancer patient, one Crohn’s disease patient and three unknown health status; alongside ‘non-gut’ reference strain SC5314) was profiled for virulence traits. C. albicans strains were profiled for biofilm formation (crystal violet staining), hyphal formation (microscopy) and cytotoxicity (lactate dehydrogenase release; during coculture with confluent Caco-2 gut epithelial cells). Additionally, the ability of C. albicans strains to translocate across epithelial cell layers was assessed during coculture with differentiated Caco-2 monolayers on Transwell™ permeable supports. Common virulence traits of hyphal formation and cytotoxicity were reduced in gut C. albicanscompared to non-gut reference strain SC5314, while biofilm formation compared to SC5314 was variable. Translocation assays revealed that gut strains of C. albicans are capable of translocating across Caco-2 epithelial cell layers in greater numbers than non-gut reference strain SC5314, with the highest levels of translocation observed from a Crohn’s disease isolate. These insights suggest that gut-specific adaptations may influence luminal escape and pathogenicity of C. albicans.

Preventive Effects of Thinned Apple Extracts on TNF-α-Induced Intestinal Tight Junction Dysfunction in Caco-2 Cells through Myosin Light Chain Kinase Suppression.

Inflammatory bowel disease (IBD) is associated with intestinal epithelial barrier dysfunction and elevation of proinflammatory cytokines such as TNF-α. Tight junctions (TJ) control the paracellular barrier of the gut. Thinned apples are an indispensable horticultural agro-waste for apple cultivation, but are disposed by most farmers. This study aimed to elucidate the preventive effect of thinned apple extracts (TAE) on the intestinal epithelial barrier dysfunction induced by TNF-α treatment in Caco-2 cells. The differentiated Caco-2 monolayers were pre-treated with mature apple extract (MAE) and TAE for 1 h and then incubated with 100 ng/mL TNF-α for 24 h. The TJ integrity was estimated by measuring the value of transepithelial electrical resistance (TEER) and the flux of fluorescein isothiocyanate-dextran through paracellular transport. TAE had a better protective effect on the intestinal epithelial barrier than MAE did. Western blot results showed that TAE pre-retreatment elevated TJ protein levels such as claudin-1, -4, and -5. Moreover, TAE inhibited the interaction between zonula occludens proteins (ZO)-1 and occludin by reducing the tyrosine phosphorylation of ZO-1. The mechanisms underlying TAE-mediated attenuation of TNF-α-induced TJ disruption included suppression of myosin light chain kinase and NF-κB p65 protein levels. Therefore, thinned apples could be a sustainable ingredient for functional foods to prevent IBD.

Investigation of eight cellulose nanomaterials’ impact on Differentiated Caco-2 monolayer integrity and cytotoxicity

The potential applications of cellulose nanomaterials (CNMs) as food additives or in food packaging, present a possible source of human ingestion. While micron- and macro-scale cellulose products are classified as Generally Regarded As Safe, the safety of ingested nano-scale cellulose is largely unknown.Using fully differentiated Caco-2 cells, the perturbation of intestinal barrier function and cytotoxicity was investigated for four nanocellulose crystals (CNCs) and four nanocellulose fibrils (CNFs) following 24 h of exposure at 50 μg/mL. Scanning electron microscope showed some aggregation of both CNCs and CNFs. X-ray photoelectron spectroscopy analyses showed that carbon and oxygen were the main elements. The zeta-potential for CNMs formulated in cell culture medium showed a negative surface charge. Two CNMs increased cell membrane permeability and three CNMs decreased the cell metabolic activity. While three CNMs lead to cytotoxic responses, no changes in apparent permeability coefficient (Papp) for dextran or tight junction integrity were found.Our results show that three CNMs induce cytotoxicity in differentiated Caco-2 cells, demonstrating the need to understand the role of size and shape. The interaction between CNMs and the intestinal epithelium needs to be evaluated to understand potential intestinal barrier dysfunction and resulting health implications following CNM ingestion.

Enteropathogenic infections modulate intestinal serotonin transporter (SERT) function by activating Toll-like receptor 2 (TLR-2) in Crohn\u2019s disease

Serotonin (5-hydroxytryptamine [5-HT]) is an intestinal neuromodulator that regulates several essential enteric physiological functions such as absorption or secretion of fluids, and peristaltic reflexes. Availability of the intestinal 5-HT is dependent on serotonin transporter (SERT), which uptakes 5-HT and facilitates its degradation. Interestingly, Toll-like receptor 2 (TLR-2) is co-localized with 5-HT, which suggests a possible impact of neuroendocrine cells in the inflammatory response through TLR-2 activation. Serum 5-HT levels were measured in 80 Crohn’s disease (CD) patients and 40 healthy control subjects. Additionally, fully differentiated Caco-2 monolayers were infected with Mycobacteria paratuberculosis (MAP), L. monocytogenes, or M. smegmatis in the presence of exogenous 5-HT at different concentrations. Cells were subsequently harvested and used for measuring SERT activity, RNA isolation followed by RT-PCR, protein quantification, and tissue damage markers (DHE, LDH, GSH and MDA). TLR-2 intracellular signaling pathways were assessed by pre-incubating Caco-2 monolayers with selective blockers of ERK, cAMP/PKA, p38 MAPK, and 5-HT3 receptors. MAP-infected CD patients (N = 40) had higher serum 5-HT levels (462.95 ± 8.55 ng/mL, N = 40) than those without MAP infection (385.33 ± 10.3 ng/mL, N = 40). TLR-2 activation by enteropathogenic bacteria inhibited SERT activity in the presence of exogenous 5-HT by up to 50%. These effects were increasing gradually over 72 h, and MAP infection had the greatest effect on SERT inhibition when cells were exposed to 5-HT in a concentration dependent manner. Additionally, inhibition of SERT activity was accompanied with higher levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-8) and oxidative stress markers (DHE, LDH and MDA), whereas SERT expression and protein level were downregulated. Consequently, inhibition of TLR-2 and p38 MAPK signaling or blocking 5-HT3 receptors restored SERT activity and reduced the production of pro-inflammatory cytokines, as reflected by the downregulation of oxidative stress and tissue damage markers. The involvement of TLR-2 in the intestinal pathology might be concluded not only from its innate immune role, but also from its effect on modulating the intestinal serotonergic response. Ultimately, regulating the intestinal serotonergic system can be therapeutically exploited to mitigate other enteropathogenic infections, which will help in understanding the gut-microbiome-brain connection.

S928 Enteropathogenic Infections Modulate Intestinal Serotonin Transporter (SERT) Function by Activating Toll-Like Receptor 2 (TLR-2) in Crohn’s Disease

Introduction: Availability of intestinal serotonin (5-HT) is dependent on serotonin transporter (SERT), which uptakes 5-HT and facilitates its degradation. Interestingly, Toll-like receptor 2 (TLR-2) is co-localized with 5-HT, which suggests a possible impact of neuroendocrine cells in the inflammatory response through TLR-2 activation. Whether modulating SERT activity and intestinal 5-HT accumulation contributes to tissue injury caused by enteropathogenic infections is unknown. Methods: Serum 5-HT levels were measured in 80 CD patients and 40 healthy control subjects. Additionally, fully differentiated Caco-2 monolayers were infected with Mycobacteria paratuberculosis (MAP), L. monocytogenes or M. smegmatis in the presence of exogenous 5-HT at different concentrations. Cells were harvested and used for measuring SERT activity, RNA isolation followed by RT-PCR, protein quantification, and tissue damage markers (DHE, LDH, GSH and MDA). TLR-2 intracellular signaling pathways were assessed by pre-incubating Caco-2 monolayers with selective blockers of ERK, cAMP/PKA, p38 MAPK, and 5-HT3 receptors. Results: Serum 5-HT levels were significantly higher among CD patients in comparison to healthy subjects [424.14 ± 9.42 ng/mL vs 211.69 ± 8.11 ng/mL, respectively]. TLR-2 activation by enteropathogenic bacteria inhibited SERT activity in the presence of exogenous 5-HT by up to 50%. These effects were increasing gradually over 72 hours, and MAP infection had the greatest effect on SERT inhibition when cells were exposed to 5-HT in a concentration dependent manner. Additionally, inhibition of SERT activity was accompanied with higher level of pro-inflammatory cytokines (TNF-α, IL-6, IL-8) and oxidative stress markers (DHE, LDH and MDA), whereas SERT expression and protein level were downregulated. Consequently, inhibition of TLR-2 and p38 MAPK pathways or blocking 5-HT3 restored SERT activity and reduced the production of pro-inflammatory cytokines by about 2 folds, which was reflected on downregulating oxidative stress and tissue damage markers. Conclusion: The involvement of TLR-2 in intestinal pathology might be concluded not only from its innate immune role, but also from its effect on modulating the intestinal serotonergic response. Ultimately, regulating the intestinal serotonergic system could provide therapeutic approaches in enteropathogenic infections, which will help in understanding the gut-microbiome-brain connection.Figure 1.: Proposed mechanism for increasing intestinal and serum 5-HT among CD patients. Enterochromaffin cells release 5-HT into the intestinal lumen. Under normal conditions, SERT activity is able to reuptake 5-HT back into the intracellular compartment, which regulates intestinal 5-HT availability. However, enteropathogenic infections cause inflammation by activating TLR-2, which reduces SERT expression and function, leading to accumulation of 5-HT and disruption of the intestinal tight junctions. This results in paracellular movement of 5-HT across the intestinal epithelium to the blood vessels, where it can cause further systemic complications.

Influence of cellulose nanocrystals (CNC) on permeation through intestinal monolayer and mucus model in vitro

Cellulose nanocrystals (CNC) as a novel ingredient in foods and pharmaceuticals still lacks the safety and functionality information. We aimed to assess the absorption of CNC in small intestine and the effect on cell viability. In the second part, the impact of CNC on substance permeation through mucus layer, including the potential functionality in improving high blood cholesterol, was tested. No noticeable amount of CNC was found to penetrate through differentiated Caco-2 monolayer and in vitro mucus layer, and CNC had low toxicity on Caco-2 cell viability up to 10 mg/mL. CNC at 2 % (w/w) may affect the permeability of the mucus layer and larger molecules are more easily influenced. CNC may also alleviate hypercholesteremia by increasing viscosity of digesta, adsorbing cholesterol, and decreasing bile acids permeation. The results suggest CNC may not penetrate the small intestinal lining and may be used as a functional supplement.

Evaluation of inhibitors of intestinal UDP-glucuronosyltransferases 1A8 and 1A10 using raloxifene as a substrate in Caco-2 cells: Studies with four flavonoids of Scutellaria baicalensis

UDP glucuronosyltransferases (UGTs) of the gastrointestinal tract play a crucial role in protection against the toxic effects of xenobiotics in the environment. UGTs such as UGT1A8 and UGT1A10 are predominantly expressed in gastrointestinal tissues. In this study, we examined the phase II metabolism of raloxifene in differentiated Caco-2 monolayers by inducing UGT1A8 and UGT1A10 expression in these cells. The present study evaluated the following four flavonoids of Scutellaria baicalensis as model herbal compounds: scutellarein, salvigenin, baicalein, and wogonin. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The transepithelial electrical resistance values for the individual compounds were comparable regardless of whether they were measured individually. Salvigenin significantly inhibited UGT1A8 and UGT1A10 activities in a concentration-dependent manner. All individual compounds except scutellarein inhibited UGT1A8 and UGT1A10 activity at a concentration of 100 μM. In addition, all individual flavonoids at 100 μM, except wogonin, significantly increased the amount of raloxifene in the basolateral chambers. The positive control, canagliflozin, significantly inhibited both UGT1A8 and UGT1A10 activities. These findings suggest that the Caco-2 assay can be utilized for identifying UGT1A8 and UGT1A10 inhibitors and indicate the potential of salvigenin for enhancing the pharmacological effects of UGT substrate drugs.

Investigation of twenty metal, metal oxide, and metal sulfide nanoparticles' impact on differentiated Caco-2 monolayer integrity

The use of engineered nanomaterials (ENMs) in foods and consumer products is rising, increasing the potential for unintentional ingestion. While the cytotoxicity of many ENMs has been investigated, less attention has been given to adverse impact on the intestinal barrier integrity. Chronical disruption of gastrointestinal integrity can have far reaching health implications.Using fully differentiated Caco-2 cells, the perturbation of intestinal barrier function and cytotoxicity were investigated for 20 metal, metal oxide, and metal sulfide ENMs. Caco-2 cells were exposed to 50 μg/mL ENMs for 24 h. ENM formulations were characterized at 0 and 24 h, and In Vitro Sedimentation, Diffusion and Dosimetry Modeling was applied to calculate the effective dose of exposure during 24 h. The apparent permeability coefficient (Papp) was determined for fluorescent labeled dextran (3000 Da) and tight junction integrity was evaluated by immunofluorescence microscopy. Cytotoxicity was investigated by determining lactate dehydrogenase release (LDH) and cell metabolic activity (tetrazolium based MTS) assays.Four ENMs led to significantly increased Papp, (15.8% w/w% Ag-SiO2 nanoparticle (NP), 60 nm CdS NP, 100 nm V2O5 flakes, and 50 nm ZnO NP), while one ENM (20 nm MgO NP) decreased Papp. With the exception of CdS NP, significantly increased Papp was not connected with cell cytotoxicity. The calculated effective dose concentration was not correlated with increased Papp.Our results illustrate that while many metal, metal oxide, and metal sulfide ENMs do not adversely affect monolayer integrity or induce cytotoxicity in differentiated Caco-2 cells, a subset of ENMs may compromise the intestinal integrity. This study demonstrated the use of differentiated Caco-2 monolayer and Papp as an endpoint to identify and prioritize ENMs that should be investigated further. The interaction between ENMs and the intestinal epithelium needs to be evaluated to understand potential intestinal barrier dysfunction and resulting health implications.

The Influence of Heat-Killed Enterococcus faecium BGPAS1-3 on the Tight Junction Protein Expression and Immune Function in Differentiated Caco-2 Cells Infected With Listeria monocytogenes ATCC 19111.

Listeria monocytogenes, the common foodborne pathogenic bacteria species, compromises the intestinal epithelial barrier, leading to development of the listeriosis, a severe disease especially among immunocompromised individuals. L. monocytogenes infection usually requires antibiotic treatment, however, excessive use of antibiotics promotes emergence of antibiotic resistance and the destruction of gut microbiota. Probiotics, including lactic acid bacteria (LAB), have been repeatedly proven as an alternative approach for the treatment of various infections. We have analyzed the potential of Enterococcus faecium BGPAS1-3, a dairy isolate exhibiting strong direct antilisterial effect, to modulate the response of differentiated Caco-2 intestinal epithelial cells to L. monocytogenes ATCC 19111 infection. We showed that the molecule with antilisterial effect is a bacterial cell-wall protein that is highly resistant to the high-temperature treatment. When we tested the antilisterial potential of heat-killed BGPAS1-3, we found that it could prevent tight junction disruption in differentiated Caco-2 monolayer infected with L. monocytogenes ATCC 19111, induce antilisterial host response mechanisms, and stimulate the production of protective TGF-β in intestinal epithelial cells. We also showed that the modulation of MyD88 dependent TLR2 and TLR4 pathways by BGPAS1-3 are involved in host response against L. monocytogenes ATCC 19111. Since heat-killed BGPAS1-3 possess strong antilisterial effects, such postbiotic could be used as a controllable and safe therapeutic.

Expression and localization of the major receptor of vasoactive intestinal peptide (VPAC1) along the length of the intestine

Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide with a broad array of physiological functions in the nervous system and peripheral organs including the intestine. This peptide exerts its action via binding to VPAC1, VPAC2 and PAC1, which are G‐protein coupled receptors (GPCR). VPAC1 is the major receptor constitutively expressed in many cell types and is responsible for majority of VIP's biological activity. However, the distribution of VPAC1 along the length of the gastrointestinal tract and its sub cellular localization in intestinal epithelial cells has not been fully defined. Therefore, the current studies were undertaken to determine VPAC1 distribution and localization so that VIP based therapies can be targeted to specific regions of the intestine.MethodsReal time PCR, immunoblotting and immuno‐fluorescence staining were used to determine the mRNA, protein and cellular localization of VPAC1 respectively. Intestinal tissues from male C57Bl/6 mice (6–8 weeks) and human tissues as well as colonic adenocarcinoma cell lines were used. The results indicated that the mRNA levels of VPAC1 in mouse intestine were highest in colon followed by jejunum and ileum. In parallel, the VPAC1 protein levels were higher in the mouse colon (proximal > distal) followed by the jejunum and ileum. Immuno‐fluorescence studies in mouse colonic tissue sections demonstrated that the receptor was specifically localized to the luminal surface as evident by co‐localization of the VPAC1 protein with the apical marker villin but not with the basolateral marker sodium potassium ATPase. In the human intestine, VPAC1 mRNA expression exhibited an expression pattern similar to mouse intestine (colon > jejunum> ileum). Furthermore, in human colon, VPAC1 predominantly showed apical localization as observed by immuno fluorescence staining. Western analysis demonstrated almost equal protein levels of VPAC1 in human adenocarcinoma cell lines e.g. Caco2, T84, HT‐29 and SKC015 cells. The luminal localization was further confirmed by relatively high apical staining for VPAC1 in differentiated Caco2 monolayers. Our current data showed differential expression of VPAC1 along the length of intestine and demonstrated its localization on the luminal membrane of colonocytes. The physiological relevance of this expression and apical localization of VPAC1 remains elusive. We speculate that apical localization of VPAC1 in intestinal epithelial cells has physiological relevance in recognizing secreted peptides in the intestinal lumen and supports the potential therapeutic use of VIP nanomedicine via oral route to target gut inflammation.Characters with spaces: 2901Support or Funding InformationSupported by NIDDK and VA Merit

Intestinal transport and absorption of bioactive phenolic compounds from a chemically characterized aqueous extract of Athrixia phylicoides

Ethnopharmacological relevanceAthrixia phylicoides, popularly known as “bush tea”, is an indigenous aromatic shrub found in mountainous and grassland areas of the northern and eastern parts of southern Africa. The plant is traditionally used for the treatment of several ailments, including coughing, treating infected wounds, treating boils and sore throat, hypertension and heart disease. Potential anti-diabetic effects have also been demonstrated in vitro. Aim of the studyTo investigate the intestinal transport of prominent phenolic constituents, across a fully differentiated Caco-2 monolayer, using a characterized aqueous extract of A. phylicoides, previously shown to have bioactivity. Materials and methodsHPLC-DAD and LC/MS analyses were used to identify the major phenolic compounds within the extract. Intestinal transport of the phenolic compounds was assessed using a differentiated Caco-2 monolayer model in order to predict bioavailability and identify metabolite formation. Rate of transport, efflux and percentage cross-over were calculated for the respective phenolic compounds. ResultsNine prominent compounds, present in the aqueous extract of A. phylicoides, were identified. Of these, three phenolic acids (protocatechuic acid, caffeic acid and para-coumaric acid), crossed the Caco-2 cell monolayer in significant amounts, with Papp values of 4.52, 4.35 (×10−6cm/s) and 2.38 (×10−5cm/s), respectively. para-Coumaric acid was shown to have the highest predicted bioavailability. ConclusionsPara-Coumaric acid, identified for the first time in A. phylicoides, was shown to have the highest predicted bioavailability suggesting that it could play a major role in the bioactivity of A. phylicoides.

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

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

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

BackgroundQuantum dots (QDs) have great potential as fluorescent labels but cytotoxicity relating to extra- and intracellular degradation in biological systems has to be addressed prior to biomedical applications. In this study, human intestinal cells (Caco-2) grown on transwell membranes were used to study penetration depth, intracellular localization, translocation and cytotoxicity of CdSe/ZnS QDs with amino and carboxyl surface modifications. The focus of this study was to compare the penetration depth of QDs in differentiated vs undifferentiated cells using confocal microscopy and image processing.ResultsCaco-2 cells were exposed to QDs with amino (NH2) and carboxyl (COOH) surface groups for 3 days using a concentration of 45 µg cadmium ml−1. Image analysis of confocal/multiphoton microscopy z-stacks revealed no penetration of QDs into the cell lumen of differentiated Caco-2 cells. Interestingly, translocation of cadmium ions onto the basolateral side of differentiated monolayers was observed using high resolution inductively coupled plasma mass spectrometry (ICP-MS). Membrane damage was neither detected after short nor long term incubation in Caco-2 cells. On the other hand, intracellular localization of QDs after exposure to undifferentiated cells was observed and QDs were partially located within lysosomes.ConclusionsIn differentiated Caco-2 monolayers, representing a model for small intestinal enterocytes, no penetration of amino and carboxyl functionalized CdSe/ZnS QDs into the cell lumen was detected using microscopy analysis and image processing. In contrast, translocation of cadmium ions onto the basolateral side could be detected using ICP-MS. However, even after long term incubation, the integrity of the cell monolayer was not impaired and no cytotoxic effects could be detected. In undifferentiated Caco-2 cells, both QD modifications could be found in the cell lumen. Only to some extend, QDs were localized in endosomes or lysosomes in these cells. The results indicate that the differentiation status of Caco-2 cells is an important factor in internalization and localization studies using Caco-2 cells. Furthermore, a combination of microscopy analysis and sensitive detection techniques like ICP-MS are necessary for studying the interaction of cadmium containing QDs with cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0222-9) contains supplementary material, which is available to authorized users.

Cell uptake and oral absorption of titanium dioxide nanoparticles

Large efforts are invested on the development of in vitro tests to evaluate nanomaterial (NM) toxicity. In order to assess the relevance of the adverse effects identified in in vitro toxicity tests a thorough understanding of the biokinetics of NMs is critical. We used different in vitro and in vivo test methods to evaluate cell uptake and oral absorption of titanium dioxide nanoparticles (TiO2 NPs). These NPs were readily uptaken by A549 cells (carcinomic human alveolar basal epithelial cells) in vitro. Such rapid uptake contrasted with a very low oral absorption in a differentiated Caco-2 monolayer system (human epithelial colorectal adenocarcinoma cells) and after oral gavage administration to rats. In this oral study, no significant increase in the levels of titanium was recorded by ICP-MS in any of the tissues evaluated (including among other: small intestine, Peyer's patches, mesenteric lymph nodes, liver, and spleen). No NPs were observed by TEM in sections of the small intestine, except for several particles in the cytoplasm of a cell from a Peyer's Patch area. The observation of NPs in Peyer's Patch suggests that the Caco-2 monolayer system is likely to underestimate the potential for oral absorption of NPs and that the model could be improved by including M-cells in co-culture.

Attenuation of glucose transport across Caco‐2 cell monolayers by a polyphenol‐rich herbal extract: Interactions with SGLT1 and GLUT2 transporters

Previous studies have indicated that secondary plant metabolites may modulate glucose absorption in the small intestine. We have characterized a polyphenol-rich herbal extract and its potential intestinal metabolites by LC-MS(2) and investigated the inhibition of glucose transporters SGLT1 and GLUT2 using the well-characterized Caco-2 intestinal model. Differentiated Caco-2 monolayers were incubated with an extract of a mixture of herbs and spices. Glucose transport under sodium-dependent and sodium-free conditions was determined by radiochemical detection of D-[U-(14) C]-glucose. A 54% decrease in transport was observed compared to control. Using sodium-dependent and sodium-free conditions, we demonstrate that the inhibition of GLUT2 was greater than SGLT1. Glycosidase and esterase enzymatic hydrolysis was used to assess the impact of metabolism on the efficacy of inhibition. Glucose transport across the membrane was reduced by 70% compared to the control and was associated with significant increases in flavonoid aglycones, caffeic acid, and p-coumaric acid. These results suggest that intact and hydrolyzed polyphenols, likely to be found in the lumen after ingestion of the supplement, play an important role in the attenuation of glucose absorption and may have potentially beneficial antiglycemic effects in the body.

Anti-Inflammatory Activities of a Chinese Herbal Formula IBS-20 In Vitro and In Vivo.

Irritable bowel syndrome (IBS) is a functional bowel disorder and the etiology is not well understood. Currently there is no cure for IBS and no existing medication induces symptom relief in all patients. IBS-20 is a 20-herb Chinese medicinal formula that offers beneficial effects in patients with IBS; however, the underlying mechanisms are largely unknown. This study showed that IBS-20 potently inhibited LPS- or IFNΓ-stimulated expression of pro-inflammatory cytokines, as well as classically activated macrophage marker nitric oxide synthase 2. Similarly, IBS-20 or the component herb Coptis chinensis decreased LPS-stimulated pro-inflammatory cytokine secretion from JAWS II dendritic cells. IBS-20 or the component herbs also blocked or attenuated the IFNΓ-induced drop in transepithelial electric resistance, an index of permeability, in fully differentiated Caco-2 monolayer. Finally, the up-regulation of key inflammatory cytokines in inflamed colon from TNBS-treated mice was suppressed significantly by orally administrated IBS-20, including IFNΓ and IL-12p40. These data indicate that the anti-inflammatory activities of IBS-20 may contribute to the beneficial effects of the herbal extract in patients with IBS, providing a potential mechanism of action for IBS-20. In addition, IBS-20 may be a potential therapeutic agent against other Th1-dominant gut pathologies such as inflammatory bowel disease.

Apple extract induces increased epithelial resistance and claudin 4 expression in Caco‐2 cells

The small intestinal epithelium functions both to absorb nutrients, and to provide a barrier between the outside, luminal, world and the human body. One of the passageways across the intestinal epithelium is paracellular diffusion, which is controlled by the properties of tight junction complexes. We used a differentiated Caco-2 monolayer as a model for small intestinal epithelium to study the effect of crude apple extracts on paracellular permeability. Exposure of crude apple homogenate to the differentiated Caco-2 cells increased the paracellular resistance, determined as trans-epithelial electrical resistance (TEER). This increase was linearly related to the concentration of apple present. The TEER-enhancing effect of apple extract was due to factors mainly present in the cortex, and the induction was not inhibited by protein kinase inhibitors. Apple-induced resistance was accompanied by increased expression of several tight junction related genes, including claudin 4 (CLDN4). Crude apple extract induces a higher paracellular resistance in differentiated Caco-2 cells. Future research will determine whether these results can be extrapolated to human small intestinal epithelia.

Hepatotoxic pyrrolizidine alkaloids—Bioavailability and cellular effects on human HEPG2 cells

Purpose: 1,2-Unsaturated pyrrolizidine alkaloids (PA) belong to the most toxic compounds. These substances are found in several plants such as Asteraceae and Boraginaceae families. Acute PA poisoning via food contamination causes severe damage to the liver; long-term, sub-lethal doses may cause cumulative damage or cancer. Methods: In this study, we analyzed the toxicological effects (cytotoxicity, ATP-content, and oxidative stress) of selected PA (retrorsine, senecionine, and seneciphylline) on the hepatocarcinoma cell line HepG2 by using a bioactivating system (S9-fraction). Furthermore, we characterized the uptake of senecionine and its N-oxidated form across the intestinal barrier by using the well-characterized human Caco-2 cell Transwell-model as an in vitro system for the human gut barrier. The PA content was analyzed by LC-MS/MS. Results: The metabolic bioactivation of PA with the S9-fraction of rat liver homogenate resulted in a weak increase of cellular cytotoxicity including a slight reduction of cellular ATP-content, as well as in a weak increase of the apoptosis rate and of oxidative stress. The Caco-2 cell absorption studies revealed a fast passage of senecionine from the apical into the basolateral compartment suggesting that active transport mechanisms are involved in the uptake and excretion of the substance. In contrast, the oxidized metabolite senecionine-N-oxide did not cross the differentiated Caco-2 monolayer from the apical to the basolateral side. In addition, we observed the conversion of senecionine into the oxidated form, as well as the reduction of senecionine-N-oxide. This indicates that Caco-2 cells are able to metabolize this compound.