Disruption Of Intercellular Junctions Research Articles

Unveiling the Impact of Hemodynamics on Endothelial Inflammation-Mediated Hepatocellular Carcinoma Metastasis Using a Biomimetic Vascular Flow Model.

Hepatocellular carcinoma (HCC) hematogenous dissemination is a leading cause of HCC-related deaths. The inflammatory facilitates this process by promoting the adhesion and invasion of tumor cells in the circulatory system. But the contribution of hemodynamics to this process remains poorly understood due to the lack of a suitable vascular flow model for investigation. This study develops a vascular flow model to examine the impact of hemodynamics on endothelial inflammation-mediated HCC metastasis. This work finds the increasing shear stress will reduce the recruitment of HCC cells by disturbing adhesion forces between endothelium and HCC cells. However, this reduction will be restored by the inflammation. When applying high FSS (4-6dyn cm-2) to the inflammatory endothelium, there will be a 4.8-fold increase in HCC cell adhesions compared to normal condition. Nevertheless, the increase fold of cell adhesions is inapparent, around 1.5-fold, with low and medium FSS. This effect can be attributed to the FSS-induced upregulation of ICAM-1 and VCAM-1 of the inflammatory endothelium, which serve to strengthen cell binding forces. These findings indicate that hemodynamics plays a key role in HCC metastasis during endothelial inflammation by regulating the expression of adhesion-related factors.

Myosin VI stabilizes intercellular junctions in the testis through the LHR and MAPK signalling pathway during spermatogenesis in Eriocheir sinensis

The myosin motor protein myosin VI plays an essential role in mammalian spermatogenesis, however, the effects of myosin VI on male reproduction in Crustacea remain obscure. We identified the macromolecule es-Myosin VI in Eriocheir sinensis, and studied it by multiple methods. It co-localized with F-actin and was highly expressed in the testis. We interfered es-Myosin VI using dsRNA in vivo, an apparent decrease in spermatozoa count was detected. We also found that the MAPK signalling pathway was changed, subsequently causing disruption of intercellular junctions and damage to the functional hemolymph–testis barrier. We observed that luteinizing hormone receptor es-LHR was located within seminiferous tubules, which was different from the expression in mammals. Es-LHR could bind with es-Myosin VI in testis of E. sinensis, its localization was significantly altered when es-Myosin VI was deleted. Moreover, we obtained consistent results for the MAPK signalling pathway and spermatogenesis defects between the es-LHR and es-Myosin VI knockdown groups. In summary, our research demonstrated that knockdown of es-Myosin VI disturbed the intercellular junction and HTB function via the MAPK signalling pathway by changing the localization of es-LHR in the testis of E. sinensis, which was the potential reason for its negative impact on spermatogenesis.

Angiotensin II type 2 receptor agonist, compound 21, prevents tubular epithelial cell damage caused by renal ischemia

During ischemic acute kidney injury (AKI), loss of cytoskeletal integrity and disruption of intercellular junctions are rapid events in response to ATP depletion. Angiotensin II type 2 receptor (AT2R) is overexpressed in injury situations and its stimulation by angiotensin II (AngII) is related to beneficial renal effects. Its role on ischemic AKI has not been deeply studied. The aim of the present study was to investigate whether pretreatment with the AT2R agonist, C21, prevents ischemic renal epithelial cell injury. Studies in a model of 40 min of renal ischemia followed by 24 h of reperfusion (IR) in rats demonstrated that C21 pretreatment attenuated renal dysfunction and induced better preservation of tubular architecture. In addition, we studied the expression of Rho GTPases, RhoA and Cdc42, since they are key proteins in the regulation of the actin cytoskeleton and the stability of epithelial intercellular junctions. IR downregulated RhoA and Cdc42 abundance in rat kidneys. C21 pretreatment prevented RhoA reduction and increased Cdc42 abundance compared to controls. We also used an in vitro model of ATP depletion in MDCK cells grown on filter support. Using immunofluorescence we observed that in MDCK cells, C21 pretreatment prevented the ATP depletion-induced reduction of actin in brush border microvilli and in stress fibers. Moreover, C21 prevented membrane E-cadherin reduction, and RhoA and Cdc42 downregulation. The present study describes for the first time a renoprotective effect of the AT2R agonist, C21, against AKI, and provides evidence supporting that stimulation of AT2R triggers cytoprotective mechanisms against an ischemic event.

Cytological Differentiation of Ductal Carcinoma in Situ and Invasive Ductal Carcinoma of the Breast: A Personal View and a Literature Review

Although some find it controversial, it is possible to differentiate breast ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) using cytology only, with certain limitations. Invasiveness is the consequence of specific biological, i.e. aggressiveness potential of malignant cells, which is different with respect to the pre-existent DCIS, consequentially with different morphology. During the invasion, malignant cells go through multiple morphological changes, losing their epithelial and acquiring mesenchymal features in the fantastic process of epithelial-mesenchymal transition, which explains their morphology in cohabitation with the environment, includes the disruption of intercellular junctions, the increase of mobility and the release of the original epithelium. This mesenchymal-like phenotype supports the migration and invasion of cells, i.e. thus epithelial-mesenchymal transition ensures the tumor dissemination and metastasizing. Therefore, invasiveness can cytologically be “measured” by detecting morphological signs of increase of biological aggressiveness of malignant cells – through the change of their appearance (cytoplasm elongation in malignant squamous cells, i.e. in adenocarcinoma intracytoplasmic lumina, atypical nucleoli, coarsely clumped chromatin, eu-/parachromatin), but also with stromal parameters (disruption of the intercellular matrix, elastin fragments, capillaries endothelium) presented by tumour diathesis, fibroblast proliferation, fragments of elastoid stroma, invasion of connective and/or adipose tissue by groups and individual malignant cells. For the invasion are also very predictive tubular malignant structures, irregular angulated clusters of reduced cohesiveness, absence of benign naked nuclei, polymorph single tumour cells, less myoepithelial cells on tumour groups, fewer microcalcifications and foamy macrophages. Opposite morphological findings suggest DCIS. Even though cytologically we do not see and cannot see the basement membrane, highly likely we can predict the invasion – necessarily and always with the triple-diagnostic approach or clinical-radiological-morphological correlation to every breast lesion, in the representative well cellular sample and with good knowledge of patohistology and cytology

Endothelial to mesenchymal transition: a precursor to post-COVID-19 interstitial pulmonary fibrosis and vascular obliteration?

Endothelial to mesenchymal transition (EndMT) could lead to post-COVID-19 pulmonary fibrosis and vascular remodelling https://bit.ly/2QqSKxT

A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways.

SummaryClostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs.

Access to a main alphaherpesvirus receptor, located basolaterally in the respiratory epithelium, is masked by intercellular junctions

The respiratory epithelium of humans and animals is frequently exposed to alphaherpesviruses, originating from either external exposure or reactivation from latency. To date, the polarity of alphaherpesvirus infection in the respiratory epithelium and the role of respiratory epithelial integrity herein has not been studied. Equine herpesvirus type 1 (EHV1), a well-known member of the alphaherpesvirus family, was used to infect equine respiratory mucosal explants and primary equine respiratory epithelial cells (EREC), grown at the air-liquid interface. EHV1 binding to and infection of mucosal explants was greatly enhanced upon destruction of the respiratory epithelium integrity with EGTA or N-acetylcysteine. EHV1 preferentially bound to and entered EREC at basolateral cell surfaces. Restriction of infection via apical inoculation was overcome by disruption of intercellular junctions. Finally, basolateral but not apical EHV1 infection of EREC was dependent on cellular N-linked glycans. Overall, our findings demonstrate that integrity of the respiratory epithelium is crucial in the host’s innate defence against primary alphaherpesvirus infections. In addition, by targeting a basolaterally located receptor in the respiratory epithelium, alphaherpesviruses have generated a strategy to efficiently escape from host defence mechanisms during reactivation from latency.

Leptospirosis in humans.

Leptospirosis is a widespread and potentially fatal zoonosis that is endemic in many tropical regions and causes large epidemics after heavy rainfall and flooding. Infection results from direct or indirect exposure to infected reservoir host animals that carry the pathogen in their renal tubules and shed pathogenic leptospires in their urine. Although many wild and domestic animals can serve as reservoir hosts, the brown rat (Rattus norvegicus) is the most important source of human infections. Individuals living in urban slum environments characterized by inadequate sanitation and poor housing are at high risk of rat exposure and leptospirosis. The global burden of leptospirosis is expected to rise with demographic shifts that favor increases in the number of urban poor in tropical regions subject to worsening storms and urban flooding due to climate change. Data emerging from prospective surveillance studies suggest that most human leptospiral infections in endemic areas are mild or asymptomatic. Development of more severe outcomes likely depends on three factors: epidemiological conditions, host susceptibility, and pathogen virulence (Fig. 1). Mortality increases with age, particularly in patients older than 60 years of age. High levels of bacteremia are associated with poor clinical outcomes and, based on animal model and in vitro studies, are related in part to poor recognition of leptospiral LPS by human TLR4. Patients with severe leptospirosis experience a cytokine storm characterized by high levels of IL-6, TNF-alpha, and IL-10. Patients with the HLA DQ6 allele are at higher risk of disease, suggesting a role for lymphocyte stimulation by a leptospiral superantigen. Leptospirosis typically presents as a nonspecific, acute febrile illness characterized by fever, myalgia, and headache and may be confused with other entities such as influenza and dengue fever. Newer diagnostic methods facilitate early diagnosis and antibiotic treatment. Patients progressing to multisystem organ failure have widespread hematogenous dissemination of pathogens. Nonoliguric (high output) renal dysfunction should be supported with fluids and electrolytes. When oliguric renal failure occurs, prompt initiation of dialysis can be life saving. Elevated bilirubin levels are due to hepatocellular damage and disruption of intercellular junctions between hepatocytes, resulting in leaking of bilirubin out of bile caniliculi. Hemorrhagic complications are common and are associated with coagulation abnormalities. Severe pulmonary hemorrhage syndrome due to extensive alveolar hemorrhage has a fatality rate of >50 %. Readers are referred to earlier, excellent summaries related to this subject (Adler and de la Peña-Moctezuma 2010; Bharti et al. 2003; Hartskeerl et al. 2011; Ko et al. 2009; Levett 2001; McBride et al. 2005).

Abstract W MP40: Pyruvate Protects Post-Hypoxic Neuronal Cells and a Blood Brain Barrier Model From rtPA Toxicity

Recombinant tissue plasminogen activator (rtPA) is the only FDA-approved treatment for ischemic stroke. However, rtPA’s therapeutic window is limited to 4.5 h after stroke onset due to hemorrhagic transformation and neurotoxicity. Here, we demonstrated that the intermediary metabolite pyruvate protects neuronal cells and a blood brain barrier (BBB) model from delayed rtPA toxicity in an in vitro oxygen-glucose deprivation (OGD, 0.5% O 2 )-reoxygenation model of ischemic stroke. After 3 or 6 h OGD, neuronal cells were reoxygenated with 11 mM glucose ± 8 mM pyruvate and/or 10 μg/ml rtPA. Cellular viability, reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP2) activity, and cellular contents of NADPH, NADP + , ATP, MMP2, tissue inhibitor of metalloproteinase-2 (TIMP2), and phosphor-activation of anti-apoptotic p70s6 kinase, Akt and Erk were measured. Pyruvate treatment after 3 h OGD decreased cell death by 80% in the absence (P < 0.01) and 64% in the presence (P < 0.01) of rtPA. After 6 h OGD, rtPA exacerbated cell death; pyruvate dampened this effect. Three hours OGD and 4 h reoxygenation + rtPA increased ROS formation by 50%. Pyruvate prevented this ROS formation and doubled antioxidant NADPH/NADP + ratio and ATP content. In the BBB model, 3 h OGD and 24 h reoxygenation increased FITC-dextran leakage, indicating disruption of intercellular junctions. Although rtPA exacerbated this effect, pyruvate prevented it while sharply lowering MMP2/TIMP2 ratio and increasing phosphorylation of p70s6 kinase, Akt and Erk. Pyruvate protects neuronal cells and BBB tight junctions from OGD-reoxygenation and rtPA toxicity while reducing ROS and activating anti-apoptotic signaling. These results support the use of pyruvate as an adjuvant to dampen rtPA’s side effects, thereby extending rtPA’s therapeutic window.

Pyruvate minimizes rtPA toxicity from in vitro oxygen-glucose deprivation and reoxygenation

Clinical application of recombinant tissue plasminogen activator (rtPA) for stroke is limited by hemorrhagic transformation, which narrows rtPA’s therapeutic window. In addition, mounting evidence indicates that rtPA is potentially neurotoxic if it traverses a compromised blood brain barrier. Here, we demonstrated that pyruvate protects cultured HT22 neuronal and primary microvascular endothelial cells co-cultured with primary astrocytes from oxygen glucose deprivation (OGD)/reoxygenation stress and rtPA cytotoxicity. After 3 or 6h OGD, cells were reoxygenated with 11mmol/L glucose±pyruvate (8mmol/L) and/or rtPA (10µg/ml). Measured variables included cellular viability (calcein AM and annexin-V/propidium iodide), reactive oxygen species (ROS; mitosox red and 2′,7′-dichlorofluorescein diacetate), NADPH, NADP+ and ATP contents (spectrophotometry), matrix metalloproteinase-2 (MMP2) activities (gelatin zymography), and cellular contents of MMP2, tissue inhibitor of metalloproteinase-2 (TIMP2), and phosphor-activation of anti-apoptotic p70s6 kinase, Akt and Erk (immunoblot). Pyruvate prevented the loss of HT22 cells after 3h OGD±rtPA. After 6h OGD, rtPA sharply lowered cell viability; pyruvate dampened this effect. Three hours OGD and 4h reoxygenation with rtPA increased ROS formation by about 50%. Pyruvate prevented this ROS formation and doubled cellular NADPH/NADP+ ratio and ATP content. In endothelial cell monolayers, 3h OGD and 24h reoxygenation increased FITC-dextran leakage, indicating disruption of intercellular junctions. Although rtPA exacerbated this effect, pyruvate prevented it while sharply lowering MMP2/TIMP2 ratio and increasing phosphorylation of p70s6 kinase, Akt and Erk. Pyruvate protects neuronal cells and microvascular endothelium from hypoxia-reoxygenation and cytotoxic action of rtPA while reducing ROS and activating anti-apoptotic signaling. These results support the proposed use of pyruvate as an adjuvant to dampen the side effects of rtPA treatment, thereby extending rtPA’s therapeutic window.

Overexpression of CD157 Contributes to Epithelial Ovarian Cancer Progression by Promoting Mesenchymal Differentiation

Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for improving patients’ survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to anoikis, increased anchorage-independent growth, cell motility and mesothelial invasion. Conversely, knockdown of CD157 in OV-90 and OC314 cells reverts the mesenchymal phenotype and reduces the cells’ migratory potential. Transcriptome profiling analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and response to stress). Together, these findings implicate CD157 in the progression of EOC to metastatic disease and suggest that CD157 may represent a valuable therapeutic target.

P29. Regulation of TGF-β1 in intercellular junctions in epithelial ovarian carcinomas

Epithelial ovarian cancer (EOC) commonly thought to arise from ovarian surface epithelia (OSE) covering the ovaries is the fifth most common cause of cancer related death in females in the Western world. It is the most lethal form of all gynecological malignancy due to the diagnosis at late stages. The pathogenesis of EOC and mechanisms underlying this event remain poorly defined. Epithelial–mesenchymal transition (EMT), a process with loss of polarity and disruption of intercellular junctions, is vital for embryonic morphogenesis as well as cancer metastasis. We have shown that TGF-β1 can induce EMT in OSE in vitro . In this study, we investigated whether TGF-β1 induces EMT in the cultured ovarian cancer cell line, OVCAR-3. We studied phenotype and trans-epithelial resistance in a filter culture insert system. Regulation of TGF-β1 in cell–cell adhesion was examined by analyzing the expression of tight junction proteins, occludin, claudin-1, -3, -4 and -7, and adherens junction proteins N-cadherin and E-cadherin using Western blotting and their mRNA expression by qPCR. We did not observe any cell phenotypic difference between TGF-β1 treated and untreated OVCAR-3. However, we did find a decrease of trans-epithelial resistance in TGF-β1 treated cells concomitant with downregulated protein and/or RNA expressions of E-cadherin, occludin as well as claudin-3, and -4, -7 whereas upregulated N-cadherin and claudin 1 in TGF-β1 treated cells. Our results suggest that TGF-β1 can modulate the formation of tight junction and adherens junction and the ion-barrier function of tight junction in OVCAR-3 without transition to a mesenchymal phenotype.

C-Jun N-Terminal kinase mediates disassembly of apical junctions in model intestinal epithelia

Dynamic remodeling of intercellular junctions is a critical determinant of epithelial barrier function in both physiological and pathophysiological states. While the disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ) has been well-described in response to pathogens and other external stressors, the role of stress-related signaling in TJ/AJ regulation remains poorly understood. The aim of this study was to define the role of stress-activated c-Jun N-terminal kinase (JNK) in disruption of intercellular junctions in model intestinal epithelia. We show that rapid AJ/TJ disassembly triggered by extracellular calcium depletion of T84 and SK-CO15 cell monolayers was accompanied by activation (phosphorylation) of JNK, and prevented by pharmacological inhibitors of JNK. The opposite process, TJ/AJ reassembly, was accelerated by JNK inhibition and suppressed by the JNK activator anisomycin. JNK1 but not JNK2 was found to colocalize with intercellular junctions, and siRNA-mediated down-regulation of JNK1 attenuated the TJ/AJ disruption caused by calcium depletion. JNK inhibition also blocked formation of characteristic contractile F-actin rings in calcium-depleted epithelial cells, suggesting that JNK regulates junctions by remodeling the actin cytoskeleton. In this role JNK acts downstream of the actin-reorganizing Rho-dependent kinase (ROCK), since ROCK inhibition abrogated JNK phosphorylation and TJ/AJ disassembly after calcium depletion. Furthermore, JNK acts upstream of F-actin-membrane linker proteins of the ERM (ezrin-radixin-moesin) family, but in a complex relationship yet to be fully elucidated. Taken together, our findings suggest a novel role for JNK in the signaling pathway that links ROCK and F-actin remodeling during disassembly of epithelial junctions.

Markedly increased intracellular lipid droplets and disruption of intercellular junctions in biopsied myocardium from a patient with arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by the progressive replacement of myocardial cells by fat and fibrous tissue. Here we describe the histopathological features of biopsied myocardium from a patient with ARVC. A large amount of adipose tissue was present in the biopsy specimen, and a group of myocardial cells were isolated as an island-like region in the adipose tissue. Electron microscopic examination of cardiomyocytes revealed a large number of intracellular lipid droplets, including some extremely large droplets. Disruptions of the plasma membrane and dissociation of intercellular junctions were associated with discharge of intracellular lipid droplets into the interstitial space. The high accumulation of intracellular lipid droplets may be involved in the pathogenesis of ARVC and may have played an important role in myocardial cell death and progressive replacement of cardiomyocytes by fatty tissue in the current case.

Breaking Through the Basement

At various stages during development, epithelial cells undergo epithelial-mesenchymal transition (EMT), becoming nonadherent and motile, a process that involves coordination of distinct events (such as loss of polarity, disruption of intercellular junctions, and degradation of the basement membrane) and that also occurs in carcinoma progression (see Levayer and Lecuit). Nakaya et al . characterized the expression patterns of various EMT markers in gastrulation-stage chick embryos and found that breakdown of the basement membrane was the earliest identifiable EMT event. Electroporation of RhoA into medial epiblast cells (mesoderm precursors) in the primitive streak interfered with their ability to undergo EMT. It led to the retention of the basement membrane marker laminin in nascent mesoderm cells and in mesoderm precursors without affecting laminin expression in lateral epiblast cells. Excess RhoA also led to the formation of aggregates of nascent mesoderm cells at the streak midline. These defects were rescued by concurrent electroporation of the Rho inhibitor C3 exoenzyme. Immunofluorescence analysis revealed RhoA in the epiblast but not the mesoderm; further, whereas RhoA was identified in both apical and basal regions of lateral epiblast cells, it was only apparent in the apical portions of medial cells. Expression of the RhoA guanine nucleotide exchange factor (GEF) Net1 was basally restricted in lateral epiblast cells and down-regulated or absent in medial epiblast cells. Antisense morpholino oligonucleotides directed against RhoA and Net1 elicited premature breakdown of laminin, as did expression of dominant-negative RhoA; moreover, the Net1 -directed morpholinos reduced basal RhoA activity in epiblast cells without affecting the activity of apical RhoA. Nocodazole, which destabilizes microtubules, promoted breakdown of the basement membrane, whereas taxol, which stabilizes them, promoted retention of the basement membrane in the medial epiblast. Misexpression of RhoA caused the retention in medial epiblast cells of a posttranslationally modified form of β-tubulin, typically found near the basal cell membrane of lateral epiblast cells but absent in medial epiblast cells. Thus, the authors propose that the decreased activity of basal RhoA and destabilization of basal microtubules lead to disruption of interactions between epithelial cells and basement membrane, and thereby trigger basement membrane breakdown, at the onset of the EMT during gastrulation. Y. Nakaya, E. W. Sukowati, Y. Wu, G. Sheng, RhoA and microtubule dynamics control cell-basement membrane interaction in EMT during gastrulation. Nat. Cell Biol . 10 , 765-775 (2008). [PubMed] R Levayer, T Lecuit, Breaking down EMT. Nat. Cell Biol . 10 , 757-759 (2008). [PubMed]

HGF attenuates thrombin‐induced endothelial permeability by Tiaml‐mediated activation of the Rac pathway and by Tiam1/Rac‐dependent inhibition of the Rho pathway

Reorganization of the endothelial cell (EC) cytoskeleton and cell adhesive complexes provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases, including asthma, sepsis, and acute respiratory distress syndrome (ARDS). We have recently described the barrier-protective effects of hepatocyte growth factor (HGF) on the human pulmonary EC. In the present study, we explored the involvement of Rac-GTPase and Rac-specific nucleotide exchange factor Tiam1 in the mechanisms of EC barrier protection by HGF. HGF protected EC monolayers from thrombin-induced hyperpermeability, disruption of intercellular junctions, and formation of stress fibers and paracellular gaps by inhibiting thrombin-induced activation of Rho GTPase, Rho association with nucleotide exchange factor p115-RhoGEF, and myosin light chain phosphorylation, which was opposed by stimulation of Rac-dependent signaling. The pharmacological Rac inhibitor or silencing RNA (siRNA) based depletion of either Rac or Tiam1 significantly attenuated HGF-induced peripheral translocation of Rac effector cortactin, cortical actin ring formation, and EC barrier enhancement. Moreover, Tiam1 knockdown using the siRNA approach, attenuated the protective effect of HGF against thrombin-induced activation of Rho signaling, monolayer disruption, and EC hyperpermeability. This study demonstrates the Tiam1/Rac-dependent mechanism of HGF-induced EC barrier protection and provides novel mechanistic insights into regulation of EC permeability via dynamic interactions between Rho- and Tiam1/Rac-mediated pathways.

Characterisation of adherens and tight junctional molecules in normal animal larynx; determining a suitable model for studying molecular abnormalities in human laryngopharyngeal reflux

Background: The disruption of intercellular junctions in the larynx is a pathological feature of laryngopharyngeal reflux (LPR). Good experimental models are necessary to gain greater insight into the molecular mechanisms...

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins.

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′,-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.

Group A Streptococcus tissue invasion by CD44-mediated cell signalling.

Streptococcus pyogenes (also known as group A Streptococcus, GAS), the agent of streptococcal sore throat and invasive soft-tissue infections, attaches to human pharyngeal or skin epithelial cells through specific recognition of its hyaluronic acid capsular polysaccharide by the hyaluronic-acid-binding protein CD44 (refs 1, 2). Because ligation of CD44 by hyaluronic acid can induce epithelial cell movement on extracellular matrix, we investigated whether molecular mimicry by the GAS hyaluronic acid capsule might induce similar cellular responses. Here we show that CD44-dependent GAS binding to polarized monolayers of human keratinocytes induced marked cytoskeletal rearrangements manifested by membrane ruffling and disruption of intercellular junctions. Transduction of the signal induced by GAS binding to CD44 on the keratinocyte surface involved Rac1 and the cytoskeleton linker protein ezrin, as well as tyrosine phosphorylation of cellular proteins. Studies of bacterial translocation in two models of human skin indicated that cell signalling triggered by interaction of the GAS capsule with CD44 opened intercellular junctions and promoted tissue penetration by GAS through a paracellular route. These results support a model of host cytoskeleton manipulation and tissue invasion by an extracellular bacterial pathogen.