Model Of Epithelial Barrier Research Articles

Functional and Molecular Characterization of the Effect of Amyloid-β42 on an in vitro Epithelial Barrier Model

Recently, the blood-brain barrier (BBB) has been pointed to as an active player in neurodegenerative disorders, albeit the actual succession of pathogenic events remains to be elucidated. Amyloid-β (Aβ) is an important pathogenic player in Alzheimer's disease, and it is cleared from the brain partly by transportation across the BBB. In this work we asked the question whether Aβ-induced alteration of tight junction (TJ) protein expression is a result of the complex in situ microenvironment of the BBB or if it can be replicated in an externalized environment, such as an in vitro epithelial barrier, where barrier property changes can be investigated without confounding factors. Therefore, we treated barrier forming MDCKI and II epithelial cells with Aβ42 and investigated TJ occludin and claudin-2 protein levels and cellular distribution through western blot and immunofluorescence. To assess barrier function, we measured transepithelial resistance (TEER) and studied cell polarity through atomic force microscopy (AFM). We found that Aβ42 cell treatment increased occludin expression and decreased claudin-2 expression. With TEER, an increase in paracellular resistance was noted, which started at 10 hours and peaked at 20 hours of Aβ42 treatment. AFM analysis demonstrated an associated morphological alteration of the cell monolayer. In conclusion, we demonstrated that Aβ42 is able to modify TJ protein expression and to functionally alter barrier properties in vitro and that this effect is not conditioned by other pathogenic Alzheimer's disease events taking place in the complex brain microenvironment.

Glucocorticoids regulate barrier function and claudin expression in intestinal epithelial cells via MKP-1

Barrier dysfunction is pivotal to the pathogenesis of inflammatory bowel diseases (IBD) and collagenous colitis. Glucocorticoids restore barrier function in Crohn's disease, but whether this reflects attenuated inflammation or an epithelial-specific action has not yet been addressed. Using filter-grown Caco-2 monolayers as an in vitro model of the intestinal epithelial barrier, we observed that glucocorticoids induced a time- and dose-dependent increase in transepithelial electrical resistance (TEER) in a glucocorticoid receptor-dependent manner without altering flux of larger solutes or changing principal tight junction architecture. This was accompanied by reduced paracellular cation flux, reduced expression of the pore-forming tight junction component claudin-2, and upregulation of the sealing tight junction protein claudin-4. In contrast, expression of occludin, claudin-1, -7, or -8 was not altered. Dexamethasone increased expression and activity of MAPK phosphatase-1 and inhibition of this phosphatase prevented the glucocorticoid-induced changes in TEER and claudin expression, whereas inhibiting p38 or MEK1/2 was not sufficient to replicate the glucocorticoid effects. Upon exposure to IFN-γ, TNF-α, or IL-1β, TEERs declined in dexamethasone-treated cells but remained consistently higher than in cells not receiving glucocorticoids. Treatment with IFN/TNF resulted in an upregulation of claudin-2 that was significantly attenuated by dexamethasone, whereas increased claudin-2 expression upon IL-1β stimulation was not affected by glucocorticoids. Taken together, barrier augmentation might represent a previously unrecognized mechanism of action, potentially contributing to the therapeutic efficacy of glucocorticoids in IBD and collagenous colitis.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro

BackgroundPlasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA).ResultsAt first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68–75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NP > chitosan DNA NP = DNA unloaded chitosan NP > control (culture medium).ConclusionsChitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Proteolytic antigens interfere with endosome/lysosome fusion in epithelial cells

The airway epithelial barrier function is important in maintaining the homeostasis in the body. A number of airway disorders are associated with the epithelial barrier dysfunction. The present study aims to elucidate a possible mechanism by which the proteolytic allergens compromise the epithelial barrier function. The airway epithelial cell line, RPMI 2650 cells (Rp cells) and kidney epithelial cell line, MDCK cells, were cultured to be monolayers and used as an in vitro epithelial barrier model. House dust mite antigen, Der P1 (Der) was used as an antigen that has the proteolytic property. The epithelial barrier permeability and transepithelial resistance (TER) were used as the indicators of epithelial barrier function. Both epithelial cell lines could endocytose Der in the culture. Some of the Der was transported across the epithelial barrier to the basal chambers of the Transwells without affecting the TER. The endocytic Der could suppress the expression of ubiquitin E3 lases A20 and further interfered with the fusion of endosome/lysosome in the epithelial cells. Mite antigen, Der, can interfere with the fusion of endosome/lysosome in epithelial cells to induce the epithelial barrier dysfunction.

Exposure of silver-nanoparticles and silver-ions to lung cells in vitro at the air-liquid interface

BackgroundDue to its antibacterial properties, silver (Ag) has been used in more consumer products than any other nanomaterial so far. Despite the promising advantages posed by using Ag-nanoparticles (NPs), their interaction with mammalian systems is currently not fully understood. An exposure route via inhalation is of primary concern for humans in an occupational setting. Aim of this study was therefore to investigate the potential adverse effects of aerosolised Ag-NPs using a human epithelial airway barrier model composed of A549, monocyte derived macrophage and dendritic cells cultured in vitro at the air-liquid interface. Cell cultures were exposed to 20 nm citrate-coated Ag-NPs with a deposition of 30 and 278 ng/cm2 respectively and incubated for 4 h and 24 h. To elucidate whether any effects of Ag-NPs are due to ionic effects, Ag-Nitrate (AgNO3) solutions were aerosolised at the same molecular mass concentrations.ResultsAgglomerates of Ag-NPs were detected at 24 h post exposure in vesicular structures inside cells but the cellular integrity was not impaired upon Ag-NP exposures. Minimal cytotoxicity, by measuring the release of lactate dehydrogenase, could only be detected following a higher concentrated AgNO3-solution. A release of pro-inflammatory markers TNF-α and IL-8 was neither observed upon Ag-NP and AgNO3 exposures as well as was not affected when cells were pre-stimulated with lipopolysaccharide (LPS). Also, an induction of mRNA expression of TNF-α and IL-8, could only be observed for the highest AgNO3 concentration alone or even significantly increased when pre-stimulated with LPS after 4 h. However, this effect disappeared after 24 h. Furthermore, oxidative stress markers (HMOX-1, SOD-1) were expressed after 4 h in a concentration dependent manner following AgNO3 exposures only.ConclusionsWith an experimental setup reflecting physiological exposure conditions in the human lung more realistic, the present study indicates that Ag-NPs do not cause adverse effects and cells were only sensitive to high Ag-ion concentrations. Chronic exposure scenarios however, are needed to reveal further insight into the fate of Ag-NPs after deposition and cell interactions.

Characterisation of H441 cells as an in vitro model of distal lung epithelial barrier

No continuously growing cell line of human distal lung epithelial phenotype and with the ability to form polarised, electrically tight monolayers has been described to date. Here, we investigated, if the human lung adenocarcinoma cell line H441 has potential to overcome this issue.Epithelial barrier properties were studied by immunocytochemistry (ICC) against ZO‐1 and measurement of RT. The expression of MDR1 and organic cation/carnitine transporters (OCT/Ns) was also investigated by ICC and immunoblot. Uptake of [14C]‐TEA and [3H]‐acetylcarnitine was carried out to assess OCT/N function. Furthermore, the effects of OCT/N modulators on organic cation uptake were studied.H441 cells formed confluent, electrically tight monolayers with peak RT values of 600–1000 Ω·cm2, after 8 days in culture. Presence of P‐gp, OCT1–3, OCTN1 and 2 was confirmed by Western blot and ICC. Uptake of organic cations was time and temperature‐dependent. In the case of [14C]‐TEA, verapamil and MPP+ most efficiently inhibited uptake, whilst amantadine and HC‐3 did not show an effect. D‐carnitine and ergothioneine significantly attenuated [3H]‐acetylcarnitine uptake into H441 cells.H441 cells have the ability to form monolayers with appreciable barrier properties. Moreover, their drug transporter expression and activity is consistent with what has been reported for human alveolar epithelial cells in primary culture.

Effects of Ethanol and Oxidized Metabolites of Linoleic Acid on Caco‐2 cell Model of Intestinal Epithelial Barrier

IntroductionThe intestinal epithelial barrier plays a critical role in host defense against luminal pathogens. Ethanol (EtOH) and dietary components may alter barrier integrity resulting in increased intestinal permeability, endotoxemia and multi‐organ pathology. The aim of the present study was to evaluate the effects of EtOH, linoleic acid (LA, a major unsaturated fatty acid in the Western diet), and its bioactive oxidized metabolites (OXLAMs) on Caco‐2 cells, an in vitro model of intestinal epithelial barrier.Materials and MethodsCaco‐2 cell monolayers were exposed to LA, OXLAMs (9‐and 13‐hydroxyoctadecadienoic acids, HODEs) and/or EtOH. Transepithelial resistance (TEER), expression of intestinal tight junction (TJ) proteins, and pro‐inflammatory response were evaluated.ResultsDecreased TEER in response to EtOH, LA, and multiple HODE isoforms was observed after 24 hours of exposure. Combined effects of EtOH and 13(S)HODE or +13HODE on TEER were greater compared to EtOH and 9(S)HODE or +9HODE. The combination of EtOH and 13(S)HODE or +13HODE resulted in greater down‐regulation of TJ proteins and increased expression of TNF‐α, IL‐1β, and IL‐6 mRNA compared to EtOH alone.ConclusionCombined exposure of EtOH and OXLAMs exacerbates disruption of intestinal barrier integrity and pro‐inflammatory response in Caco‐2 cells.

Integrin alpha v beta 6 contributes to maintaining corneal epithelial barrier function

The corneal epithelial barrier dysfunction is associated with a number of disorders of the cornea. This study aims to investigate the role of integrin alpha v beta 6 (avb6) in maintaining the corneal epithelial barrier function. In this study, the association of avb6 and endosome/lysosome in the human corneal epithelial cell line, HCE cells, was observed with immunocytochemistry. The corneal epithelial barrier function was assessed with HCE monolayers in Transwells. The results showed that avb6 was observed attaching to endosomes in HCE cells. Knockdown of avb6 resulted in disturbing the fusion of endosome/lysosome. Tumor necrosis factor (TNF)-α could bind avb6 to form a complex that interfered with the fusion of endosome/lysosome in HCE cells. With the HCE monolayer as a corneal epithelial barrier model, the knockdown of avb6 or exposure to TNF-α markedly increased the epithelial barrier permeability to a macomolecular protein, ovalbumin, in Transwell system. After passing through the deficient epithelial barrier, the ovalbumin molecules still conserved the antigenicity. We conclude that the integrin avb6 plays an important role in the fusion of endosome/lysosome in corneal epithelial cells; inhibition of avb6 results in corneal epithelial barrier dysfunction.

Cholera Toxin Suppresses Expression of Ubiquitin Editing Enzyme A20 and Enhances Transcytosis

Background: The ubiquitin editing enzyme A20 plays an important role in maintaining the homeostasis in the body Microbe-derived adjuvants are commonly used in animal models of intestinal allergy. Objective: This study aims to investigate the role of cholera toxin-induced A20 suppression in compromising intestinal barrier function. Methods: Human intestinal epithelial cells were cultured into monolayers as an in vitro epithelial barrier model. Ovalbumin (OVA) was used as a specific allergen to test the degrading capability of intestinal epithelial cells for the endocytic allergens. The fusion of endosomes and lysosomes in epithelial cells was observed by immunocytochemistry. The antigenicity of OVA was tested by T cell proliferation assay. Results: A20 was detectable in the intestinal cell lines and mouse intestinal epithelialum. A20 was required in the degradation of endocytic allergens in HT-29 cells. The allergen, OVA, could pass through A20-deficient HT-29 monolayer barrier. Exposure to microbial adjuvant, cholera toxin, suppressed the expression of A20 in HT-29 cells, which compromised the epithelial barrier function. Conclusion: The microbial product, cholera toxin, interferes with the expression of A20 in intestinal epithelial cells, which compromises the intestinal epithelial barrier function.

Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro

BackgroundIncreasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (−NH2) and carboxyl (−COOH) surface modifications.ResultsPre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures.ConclusionsThe present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies.

Clinical Saccharomyces cerevisiae isolates cannot cross the epithelial barrier in vitro

Saccharomyces cerevisiae is generally considered to be a safe organism and is essential to produce many different kinds of foods as well as being widely used as a dietary supplement. However, several isolates, which are genetically related to brewing and baking yeasts, have shown virulent traits, being able to produce human infections in immunodeficient patients. Previously it has been shown that the administration of S. cerevisiae clinical isolates can lead to systemic infections, reaching several organs in murine systems. In this work, we studied S. cerevisiae clinical isolates in an in vitro intestinal epithelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. The results showed that, in contrast to C. glabrata and C. albicans, S. cerevisiae was not able to cross the intestinal barrier. We concluded that S. cerevisiae can only perform opportunistic or passive crossings when epithelial barrier integrity is previously compromised.

Differential Targeting of the E-Cadherin/β-Catenin Complex by Gram-Positive Probiotic Lactobacilli Improves Epithelial Barrier Function

The intestinal ecosystem is balanced by dynamic interactions between resident and incoming microbes, the gastrointestinal barrier, and the mucosal immune system. However, in the context of inflammatory bowel diseases (IBD), where the integrity of the gastrointestinal barrier is compromised, resident microbes contribute to the development and perpetuation of inflammation and disease. Probiotic bacteria have been shown to exert beneficial effects, e.g., enhancing epithelial barrier integrity. However, the mechanisms underlying these beneficial effects are only poorly understood. Here, we comparatively investigated the effects of four probiotic lactobacilli, namely, Lactobacillus acidophilus, L. fermentum, L. gasseri, and L. rhamnosus, in a T84 cell epithelial barrier model. Results of DNA microarray experiments indicating that lactobacilli modulate the regulation of genes encoding in particular adherence junction proteins such as E-cadherin and β-catenin were confirmed by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, we show that epithelial barrier function is modulated by Gram-positive probiotic lactobacilli via their effect on adherence junction protein expression and complex formation. In addition, incubation with lactobacilli differentially influences the phosphorylation of adherence junction proteins and the abundance of protein kinase C (PKC) isoforms such as PKCδ that thereby positively modulates epithelial barrier function. Further insight into the underlying molecular mechanisms triggered by these probiotics might also foster the development of novel strategies for the treatment of gastrointestinal diseases (e.g., IBD).

Transport of Metal Oxide Nanoparticles Across Calu-3 Cell Monolayers Modelling the Air-Blood Barrier

Abstract As inhalation is the major exposure route for nanoparticles, the question if inhaled particles can overcome the respiratory epithelial barrier and hence enter the body is of great interest. Here, we adapted the for soluble substances well established Calu-3 in vitro air-blood barrier model to the use of nanoparticle transport testing. As the usually used filter supports hindered particle transport due to their small pore size, supports with a pore size of 3 μm had to be used. On those filters, barrier and transport characteristics of the cells were tested and culture conditions changed to obtain optimal conditions. Functionality was confirmed with transport experiments with polystyrene model particles prior to testing of industrially relevant engineered metal oxide particles. Except for CeO2 nanoparticles, no transport across the epithelial barrier model could be detected. Paracellular permeability and barrier function was not affected by any of the nanoparticles, except for ZrO2.

Biodegradable Nanoparticles Meet the Bronchial Airway Barrier: How Surface Properties Affect Their Interaction with Mucus and Epithelial Cells

Despite the wide interest raised by lung administration of nanoparticles (NPs) for the treatment of various diseases, little information is available on their effect toward the airway epithelial barrier function. In this study, the potential damage of the pulmonary epithelium upon exposure to poly(lactide-co-glycolide) (PLGA) NPs has been assessed in vitro using a Calu-3-based model of the bronchial epithelial barrier. Positively and negatively charged as well as neutral PLGA NPs were obtained by coating their surface with chitosan (CS), poloxamer (PF68), or poly(vinyl alcohol) (PVA). The role of NP surface chemistry and charge on the epithelial resistance and mucus turnover, using MUC5AC as a marker, was investigated. The interaction with mucin reduced the penetration of CS- and PVA-coated NPs, while the hydrophilic PF68-coated NPs diffused across the mucus barrier leading to a higher intracellular accumulation. Only CS-coated NPs caused a transient but reversible decrease of the trans-epithelial electrical resistance (TEER). None of the NP formulations increased MUC5AC mRNA expression or the protein levels. These in vitro results highlight the safety of PLGA NPs toward the integrity and function of the bronchial airway barrier and demonstrate the crucial role of NP surface properties to achieve a controlled and sustained delivery of drugs via the pulmonary route.

Investigating the Interaction of Cellulose Nanofibers Derived from Cotton with a Sophisticated 3D Human Lung Cell Coculture

Cellulose nanofibers are an attractive component of a broad range of nanomaterials. Their intriguing mechanical properties and low cost, as well as the renewable nature of cellulose make them an appealing alternative to carbon nanotubes (CNTs), which may pose a considerable health risk when inhaled. Little is known, however, concerning the potential toxicity of aerosolized cellulose nanofibers. Using a 3D in vitro triple cell coculture model of the human epithelial airway barrier, it was observed that cellulose nanofibers isolated from cotton (CCN) elicited a significantly (p < 0.05) lower cytotoxicity and (pro-)inflammatory response than multiwalled CNTs (MWCNTs) and crocidolite asbestos fibers (CAFs). Electron tomography analysis also revealed that the intracellular localization of CCNs is different from that of both MWCNTs and CAFs, indicating fundamental differences between each different nanofibre type in their interaction with the human lung cell coculture. Thus, the data shown in the present study highlights that not only the length and stiffness determine the potential detrimental (biological) effects of any nanofiber, but that the material used can significantly affect nanofiber-cell interactions.

The NCI-N87 cell line as a gastric epithelial barrier model for drug permeability assay

The objective of this study was to evaluate the human NCI-N87 cell line as a model for gastric permeability drug studies under pH conditions of the stomach. The optimal conditions that led NCI-N87 cells to form a typical differentiated gastric epithelial barrier were a seeding density of 2.5 × 10 5 cells/cm 2 on porous inserts and growth in serum-complemented RPMI-1640 medium until 18–27 days post-confluency. The resulting cell monolayers showed moderately high transepithelial electrical resistance (TEER) values of about 500 Ω cm 2, cells of polygonal morphology expressing E-cadherin and ZO-1 proteins at their contact surfaces, and production of mucus clusters. The monolayers withstood apical pH of 7.4 down to 3.0 with the basal pH fixed at 7.4. The apparent permeability coefficients ( P app) of model compounds were evaluated in the apical-to-basolateral and basolateral-to-apical directions under different pH gradients. The monolayers were impermeable to the integrity marker Lucifer Yellow (low P app of 0.3–1.1 × 10 −6 cm/s). The furosemide P app (0.4–1.5 × 10 −5 cm/s) were slightly dependent on pH but remained moderate. The caffeine P app (4.2–5.0 × 10 −5 cm/s) were higher and insensitive to pH changes. The NCI-N87 cell line provides a useful in vitro tool to assess gastric drug permeability and absorption under physiologic conditions prevailing in the human stomach.

Mother-to-child transmission of HTLV-1:in vitro study of HTLV-1 passage across a tight human epithelial barrier

Background Besides horizontal transmission, HTLV-1 is transmitted vertically mainly through prolonged breastfeeding. Maternal transmission via breast milk is the dominant mode of HTLV-1 spread in high endemic areas, and is inked to the presence of HTLV-1 infected cells (lymphocytes, epithelial cells..) in the milk. Infection in young childhood by such mean appears to be a major risk factor for development of ATL in adults. Materials and methods We developed an in vitro model of epithelial barrier (Caco-2 human enterocytic cell line) to assess the mode of passage of HTLV-1 through the digestive tract. Integrity of the barrier was checked by ultrastructural approach, measurement of the trans-epithelial resistance (TER), and diffusion of fluorescently labeled molecules. Results When enterocytes were co-cultured with HTLV-1infected lymphocytes, no structural modifications were detected in intercellular tight junctions. Moreover, the functional integrity of the epithelial barrier was maintained since no TER change was detected in the presence of infected lymphocytes, and passage of small fluorescent markers was unaffected. Although enterocytes were not found to be susceptible to HTLV-1 infection, free infectious HTLV-1 virions were detected in the basal compartment, and such a passage was temperaturedependent, suggesting a transcytotic mechanism of passage. When human dendritic cells were added to the basal compartment, they were found to be productively infected by HTLV-1 that had crossed the epithelial barrier.

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.

Drug permeation across intestinal epithelial cells using porous silicon nanoparticles

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

Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.