Zonula Occludens-1 Staining Research Articles

Is It Hot or Cold? Which Humid Air Is Better for Vocal Hygiene?

The purpose of this research is to determine which type of humid air should be suggested for vocal hygiene by demonstrating the effects of hot humid air and cold humid air on the vocal cord mucosa using different histological methods. Randomized controlled study. Cold or hot humid air was applied to the rats for 30 minutes/day for 10 days using a humid air machine placed in a closed glass cage. The control group did not receive any treatment and were kept in their cages under normal laboratory conditions. The animals were sacrificed and their larynxes were removed on the 11th day. Histologically, lamina propria (LP) thickness was measured by Crossman's three stain and the number of mast cells in 1 square millimeter of lamina propria was measured by toluidine blue. In immunohistochemical staining, the intensity of zonula occludens-1 (ZO-1) staining was measured using a rabbit polyclonal antibody and scored from 0 (no staining) to 3 (intense staining). One-way ANOVA and Kruskal-Wallis tests were used to compare groups. The mean LP thickness was thinner in rats exposed to cold humid air (CHA) than in the control group (P=0.012). In terms of LP thickness, other intergroup comparisons (cold vs hot and control vs hot) showed no statistically significant difference between groups (P > 0.05). The mean mast cell count did not differ between groups. The hot humid air (HHA) group had more intense ZO-1 staining than the other groups (P < 0.001). There was no difference in ZO-1 staining intensity between the control group and CHA group. HHA and CHA administration had no negative effects on inflammatory findings in the vocal cords (mast cell count or LP thickness). While HHA appears to strengthen the epithelial barrier (denser ZO-1 staining), the physiologic outcomes, such as bronchoconstriction, should be cautiously assessed.

Development of Human Gut Organoids With Resident Tissue Macrophages as a Model of Intestinal Immune Responses.

Development of Human Gut Organoids With Resident Tissue Macrophages as a Model of Intestinal Immune Responses.

Small silica nanoparticles transiently modulate the intestinal permeability by actin cytoskeleton disruption in both Caco-2 and Caco-2/HT29-MTX models.

Amorphous silica nanoparticles are widely used as pharmaceutical excipients and food additive (E551). Despite the potential human health risks of mineral nanoparticles, very few data regarding their oral toxicity are currently available. This study aims to evaluate and to understand the interactions of silica particles at 1 and 10mgmL-1 with the intestinal barrier using a Caco-2 monolayer and a Caco-2/HT29-MTX co-culture. A size- and concentration-dependent reversible increase of the paracellular permeability is identified after a short-term exposure to silica nanoparticles. Nanoparticles of 30nm induce the highest transepithelial electrical resistance drop whereas no effect is observed with 200nm particles. Additive E551 affect the Caco-2 monolayer permeability. Mucus layer reduces the permeability modulation by limiting the cellular uptake of silica. After nanoparticle exposure, tight junction expression including Zonula occludens 1 (ZO-1) and Claudin 2 is not affected, whereas the actin cytoskeleton disruption of enterocytes and the widening of ZO-1 staining bands are observed. A complete permeability recovery is concomitant with the de novo filament actin assembly and the reduction of ZO-1 bands. These findings suggest the paracellular modulation by small silica particles is directly correlated to the alteration of the ZO-actin binding strongly involved in the stability of the tight junction network.

94 Essential oils improve barrier function and attenuate inflammatory responses in porcine intestinal epithelial cells

Abstract Thymol has been known as a functional phytochemical isolated from thyme essential oils and possesses antioxidant, antimicrobial, and anti-inflammatory properties. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model using IPEC-J2 cell line was established to evaluate the inflammatory responses after thymol treatment. Cells were pre-treated with thymol for 1 h followed by LPS stimulation. Interleukin 8 (IL-8) secretion, reactive oxygen species (ROS) production, mRNA abundance of two pro-inflammatory cytokines, nutrient transporters, and tight junction proteins, transepithelial electrical resistance (TEER) and cell permeability were measured. The localization of zonula occludens-1 (ZO-1) and β-actin were also detected by immunofluorescent staining. The results showed that LPS stimulation increased IL-8 secretion, ROS production, and tumor necrosis factor alpha (TNF-α) mRNA abundance (P &amp;lt; 0.05), but the mRNA abundance of sodium-dependent glucose transporter 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1) and H+/peptide cotransporter 1 (PepT1) were decreased (P &amp;lt; 0.05). However, thymol blocked ROS production (P &amp;lt; 0.05) and tended to decrease the production of LPS-induced IL-8 secretion (P = 0.0766). The mRNA abundance of IL-8 and TNF-α was reduced by thymol pre-treatment (P &amp;lt; 0.05), but thymol was unable to improve the gene expression of nutrient transporters (P &amp;gt; 0.05). TEER was reduced and cell permeability was increased after LPS stimulation (P &amp;lt; 0.05), but these effects were attenuated by thymol pre-treatment (P &amp;lt; 0.05). Moreover, thymol boosted ZO-1 and β-actin staining in the cells, but the mRNA abundance of ZO-1 and occludin-3 was not affected by either LPS or thymol treatments. These results indicated that thymol can enhance gut barrier structure and functions by reducing ROS production and pro-inflammatory cytokine gene expression in porcine epithelial cells during inflammation. The regulation of barrier function by thymol may be at post-transcriptional or post-translational levels.

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10.

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host's alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and interferon-γ and tumor necrosis factor-α-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independently of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.

Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells; assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration

BackgroundCopper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact.MethodsUndifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 μg/cm2 Cu (equivalent to 1.95 to 250 μg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 μg/cm2 for CuO NMs, and 4.25 μg/cm2 for copper sulphate (CuSO4), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO4 on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO4 on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO4 was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (Papp); a measure of barrier permeability to CuO NMs. For all experiments, CuSO4 was used as an ionic control.ResultsCuO NMs and CuSO4 caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO4 translocated across the differentiated Caco-2 cell monolayer. CuO NM mediated IL-8 production was over 2-fold higher in undifferentiated cells. A reduction in cell viability in differentiated cells was not responsible for the lower level of cytokine production observed. Both CuO NMs and CuSO4 decreased TEER values to a similar extent, and caused tight junction dysfunction (ZO-1 staining), suggesting that barrier integrity was disrupted.ConclusionsCuO NMs and CuSO4 stimulated IL-8 production by Caco-2 cells, decreased barrier integrity and thereby increased the Papp and translocation of Cu. There was no significant enhancement in potency of the CuO NMs compared to CuSO4. Differentiated Caco-2 cells were identified as a powerful model to assess the impacts of ingested NMs on the GI tract.

Interleukin-18 induces retinal pigment epithelium degeneration in mice.

To examine the effectiveness of interleukin-18 (IL-18) on choroidal neovascularization (CNV) and retinal pigment epithelium (RPE) in humans and mice. Serum IL-18 levels in patients with wet and dry AMD who were older than 50 years were measured and compared with those of age-matched controls. In mice, laser photocoagulation was performed in the retina to induce experimental CNV, and CNV volume was measured in eyes injected with recombinant IL-18 (rIL-18) and IL-18 neutralizing antibody (nIL-18Ab) compared with those injected with control. Tube formation assay was performed on human retinal endothelial cells (HREC) with rIL-18 administration in vitro. After subretinal injection of rIL-18, fundus change in the injected eyes was evaluated; active caspase-3 level was measured in the RPE/choroid complex, and tight junction integrity in RPE was visualized by zonula occludens-1 (ZO-1) staining. Serum IL-18 levels in dry AMD patients were higher than those in control. Mouse rIL-18 or nIL-18Ab did not induce significant change in CNV volume compared with controls or change tube formation in HREC. Subretinal injection of rIL-18 induced retinal degeneration in the mice fundus; ZO-1 staining showed considerably disturbed RPE structure, and active caspase-3 expression was significantly higher after rIL-18 induction. Interleukin-18 did not show a pro- or antiangiogenic effect on mouse laser-induced CNVs (laser-CNVs), whereas it directly induced RPE cell apoptosis in the mouse eye. Our results suggested that IL-18 is associated with dry AMD, but not with wet AMD.

AMPK preserves endothelial tight junctions in the coronary microcirculation during sepsis

Purpose: Compromising endothelial cell (EC) monolayer affects vascular permeability and leads to fluid leakage. Tight junctions, and more particularly, zonula occludens 1 (ZO-1) plays a major role in maintaining vascular barrier integrity and is regulated by cytoskeletal proteins. During sepsis, endothelial barrier disruption occurs in most organs and contributes to organ dysfunction. We and others have demonstrated that the α1 isoform of AMP- activated protein kinase (α1AMPK) controls cytoskeleton organisation in various cell types including endothelial cells. Therefore, we hypothesized that α1AMPK preserves tight junctions organisation and vascular permeability during sepsis in the coronary microcirculation. Methods: In vitro, tight junctions organisation (ZO-1 staining), cytoskeleton organisation (phalloidin staining) and vascular permeability were measured in endothelial cells in culture (HCAECs). Endothelial cells were pretreated with 1mM AICAriboside (AICAr) before LPS challenge (50μg/mL O55B5). In vivo, wild-type (α1AMPK +/+) mice were treated with LPS (O55B5,10mg.kg-1) and compared to α1-AMPK knock-out animals (α1AMPK -/-). ZO-1 localisation was determined with immunostaining. C57BL6/J mice were treated with AICAr (800 mg/kg) and challenged with LPS (O55B5,10mg.kg-1). Cardiac vascular permeability was evaluated using Evans Blue dye leakage. Results: In vitro, LPS-challenged cells displayed a significant disruption of the ZO-1 linear configuration after 24 hours and exhibited a decrease in peripheral actin filaments. Gap areas appeared in the cellular monolayer exposed to LPS, unlike untreated cells for which the monolayer remained unaltered. Consequently, LPS treatment gradually increased endothelial cells monolayer permeability in a time dependent manner. AMPK activation by AICAr preserved linear pattern of ZO-1 and prevented gap areas in the monolayer. More interestingly, AMPK activation reduced LPS-induced hyperpermeability. In vivo, according to the in vitro experiments, a dramatic decrease in ZO-1 staining was observed in α1AMPK -/- hearts compared to α1AMPK +/+, after LPS challenge. This resulted in an increased Evans blue extravasation. Finally, treating C57Bl6/J mice with AICAr reduced cardiac vascular hyperpermeability. Conclusions: AMPK signalling pathway protects endothelial barrier during sepsis by preserving tight junctions organisation. Since AMPK counteracts the molecular events involved in the LPS-induced barrier disruption in coronary microcirculation, it could consequently represent a new therapeutic molecule during septic cardiopathy.

Apical vacuole formation by gastric parietal cells in primary culture: effect of low extracellular Ca2+

In primary culture, the gastric parietal cell's deeply invaginated apical membrane, seen in microscopy by phalloidin binding to F-actin (concentrated in microvilli and a subapical web), is engulfed into the cell, separated from the basolateral membrane (which then becomes the complete plasma membrane), and converted, from a lacy interconnected system of canaliculi, into several separate vacuoles. In this study, vacuolar morphology was achieved by 71% of parietal cells 8 h after typical collagenase digestion of rabbit gastric mucosa, but the tight-junctional protein zonula occludens-1 (ZO-1) was completely delocalized after ∼2 h, when cells were ready for culturing. Use of low-Ca(2+) medium (4 mM EGTA) to release cells quickly from gastric glands yielded parietal cells in which ZO-1 was seen in a small spot or ring, a localization quickly lost if these cells were then cultured in normal Ca(2+) but remaining up to 20 h if they were cultured in low Ca(2+). The cells in low Ca(2+) mostly retained, at 20 h, an intermediate morphology of many bulbous canalicular expansions ("prevacuoles"), seemingly with narrow interconnections. Histamine stimulation of 20-h cells with intermediate morphology caused colocalization of proton-pumping H-K-ATPase with canaliculi and prevacuoles but little swelling of those structures, consistent with a remaining apical pore through which secreted acid could escape. Apparent canalicular interconnections, lack of stimulated swelling, and lingering ZO-1 staining indicate inhibition of membrane fission processes that separate apical from basolateral membrane and vacuoles from each other, suggesting an important role for extracellular Ca(2+) in these, and possibly other, endocytotic processes.

Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin

ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland.

Localization of P-gp (Abcb1) and Mrp2 (Abcc2) in Freshly Isolated Rat Hepatocytes

Freshly isolated hepatocytes are widely accepted as the "gold standard" for providing reliable data on drug uptake across the sinusoidal (basolateral) membrane. However, the suitability of freshly isolated hepatocytes in suspension to assess efflux by canalicular (apical) proteins or predict biliary excretion in the intact organ is unclear. After collagenase digestion, hepatocytes rapidly lose polarity, but localization of canalicular transport proteins in the first few hours after isolation has not been well characterized. In this study, immunostaining and confocal microscopy have provided, for the first time, a detailed examination of canalicular transport protein localization in freshly isolated rat hepatocytes fixed within 1 h of isolation and in cells cultured for 1 h. Organic anion transporting polypeptide 1a1 (Oatp1a1) was expressed in all hepatocytes and distributed evenly across the basolateral membrane; there was no evidence for colocalization of Oatp1a1 with P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (Mrp2). In contrast, P-gp and Mrp2 expression was lower than Oatp1a1 and confined to junctions between adjacent cells, intracellular compartments, and "legacy" network structures at or near the cell surface. P-gp and Mrp2 staining was more predominant in regions adjacent to former canalicular spaces, identified by zonula occludens-1 staining. Functional analysis of rat hepatocytes cultured for 1 h demonstrated that the fluorescent anion and Mrp2 substrate, 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDF), accumulated in cellular compartments; compartmental accumulation of CDF was sensitive to (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571, Mrp inhibitor) and was not observed in hepatocytes isolated from Mrp2-deficient rats. Drug efflux from freshly isolated hepatocytes as an estimate of apical efflux/biliary excretion would give an inaccurate assessment of true apical elimination and, as such, should not be used to make in vivo extrapolations.

Zonula occludens‐1 and connexin 43 expression in the failing human heart

Focal disorganization of gap junctional distribution and down-regulation of the major gap junctional protein connexin 43 are typical features of myocardial remodelling in the failing human heart. Increasing evidence indicates that connexin 43 interacts with zonula-occludens-1 (ZO-1), and it has recently been shown that ZO-1 promotes the formation and growth of gap junctional plaques. In the present study, distribution patterns of ZO-1 and connexin 43 were studied in normal and in heart failure patients using double-label immunohistochemistry and confocal microscopy. ZO-1 was found to be co-localized with connexin 43 at intercalated disks. Importantly, in patients with heart failure due to dilated or ischaemic cardimyopathy, areas of diminished connexin 43 expression were characterized by a markedly reduced ZO-1 staining. Based on these data it is concluded that in patients with heart failure, down-regulation of ZO-1 matches the diminished expression levels of connexin 43, suggesting that ZO-1 plays an important role in gap junction formation and gap junction plaque stability.

Strengthening tight junctions of retinal microvascular endothelial cells by pericytes under normoxia and hypoxia involving angiopoietin-1 signal way

To determine the effects of pericytes and angiopoietin-1 on the expression of occludin and zonula occludens-1 (ZO-1) in retinal endothelial cells (ECs) under both normoxic and hypoxic conditions. Rat primary retinal microvascular ECs were cultured under normoxia or hypoxia in either absence or presence of pericytes conditioned medium (PCM). PCM was pretreated with or without angiopoietin-1 neutralizing antibody. Immuofluorescent staining, Western blot and RT-PCR were used to detect the alterations of occludin and ZO-1 expression. Under normoxia, PCM strengthened occludin and ZO-1 immunofluorescent staining at cytomembrane as well as increased their expression at both protein and mRNA level. When pretreated with angiopoietin-1 neutralizing antibody, occludin upregulation induced by PCM was significantly blocked at protein level (62%) and mRNA level (34%). Under hypoxia, the continuity of occludin and ZO-1 staining at cell boundaries was disrupted consistent with a decrease of their protein level by 31 and 27%, respectively. Also occludin and ZO-1 mRNA level decreased by 46 and 57%, respectively. PCM was observed to partially increase expression of occludin at protein and mRNA level. Angiopoietin-1 antibody slightly inhibited (16%) PCM induced occludin mRNA increase under hypoxia. Pericytes improved the integrity of endothelial barrier through inducing occludin and ZO-1 expression at protein and mRNA level under normoxia. Under hypoxia, pericytes could partially reverse occludin decrease. These protecting effects of pericytes on endothelial barrier were at least in part mediated by angiopoietin-1.

Permselective Dysfunction of Podocyte-Podocyte Contact upon Angiotensin II Unravels the Molecular Target for Renoprotective Intervention

Ameliorating the function of the glomerular barrier to circulating proteins by blocking angiotensin II (Ang II) translates into less risk of progression toward end-stage renal failure in diabetic and nondiabetic nephropathies. However, the mechanisms underlying this barrier protection are not clear. Specialized contacts between adjacent podocytes are major candidate targets, and the actin cytoskeleton is emerging as a regulatory element. Here, we present data demonstrating that Ang II induced reorganization of F-actin fibers and redistribution of zonula occludens-1 (ZO-1) that is physically associated with actin in murine podocytes. These effects were paralleled by increased albumin permeability across podocyte monolayers. The F-actin stabilizer jasplakinolide prevented both ZO-1 redistribution and albumin leakage, suggesting that actin cytoskeleton rearrangement is instrumental to podocyte permselective dysfunction induced by Ang II. Changes in both F-actin and ZO-1 patterns were confirmed in glomeruli of rat isolated perfused kidneys on short infusion of Ang II, leading to increased protein excretion. Podocyte dysfunction was mediated by Ang II type 1 receptor and was partly dependent on Src kinase-phospholipase C activation. These data demonstrate that strategies aimed at stabilizing podocyte-podocyte contacts and targeting the relevant intracellular signal transduction are crucial to renoprotection.

The primary phase of infection by pathogenic simian-human immunodeficiency virus results in disruption of the blood-brain barrier.

Using the simian-human immunodeficiency virus (SHIV), we have investigated whether the blood-brain barrier (BBB) is compromised during the early stages of infection. Five macaques were inoculated with pathogenic SHIV(50OLNV) for 2 weeks at which time macaques were anesthetized, perfused with saline, and sacrificed. The brains were removed and examined for the disruption of the blood-brain barrier by immunohistochemical staining for the plasma protein fibrinogen in the neural parenchyma. Our results indicate a disruption of the BBB in the five of five macaques inoculated with SHIV(50OLNV) for 2 weeks. Zonula occludens 1 (ZO-1), which is a marker for the tight junctions formed by brain vascular endothelial cells, was largely absent in areas that showed fibrinogen deposition in all five macaques. To determine if the BBB integrity correlated with the initial stages of infection, the brains from two macaques were analyzed that had progressed to end-stage disease following inoculation with pathogenic SHIV(50OLNV) but developed no neuropathology and from two macaques that were inoculated with a gene-deleted, nonpathogenic virus (novpuSHIV(KU-1bMC33)) for over 1 year. Our results indicate that unlike the macaques sacrificed during the acute phase of infection, immunohistochemical staining for fibrinogen in the neural parenchyma was negative and ZO-1 staining was readily detected in the endothelial cells of the blood vessels. The results of this study indicate that the transient loss of BBB integrity is a function of the high level of virus replication that occurs during the acute phase of infection and provides important information on the early stages of lentivirus neuroinvasion.

Epithelial barrier integrity during in vitro wound repair of the airway epithelium.

The surface epithelium of the airway mucosa forms a continuous barrier to a wide number of noxious substances present in the lumen. The restoration of the barrier integrity after injury represents a key issue in the defense capacity of the airway epithelium. Using an in vitro wound repair model of the airway epithelium, we investigated the dynamic of the restoration of the epithelial barrier integrity during the wound repair process. Airway epithelial cells in culture were chemically wounded by sodium hydroxide. The immunolocalization of zonula occludens 1 (ZO-1), a cytoplasmic protein associated with the tight junctions, was examined during the wound repair process. Junctional integrity was examined by analyzing the transepithelial resistance (TER) and the permeability to [3H]mannitol and by visualizing the permeability to lanthanum nitrate during 5 days after injury. Immediately after injury, we simultaneously observed a 36.7% decrease in the TER and a 74.9% rise in the permeability to [3H]mannitol. In addition, lanthanum nitrate penetrated in the intercellular spaces in the repairing areas, which was also characterized by the absence of ZO-1 staining, as opposed to nonrepairing cells. TER and [3H]mannitol flux values as well as lanthanum nitrate and ZO-1 localizations were found to be similar to those observed in confluent cultures only 1 to 2 days after complete wound closure. This study demonstrates that using our culture model, confluent airway epithelial cells form a continuous and efficient barrier with tight junctions. Epithelial integrity is affected immediately after injury and is completely restored within 1 to 2 days after wound closure. During such a period of time, the airway epithelium may remain exposed to the noxious effect of environment in vivo, which can prevent the epithelial barrier restoration by modifying tight junction formation.