Changes In Paracellular Permeability Research Articles

Colonic Epithelial Permeability to Ions Is Restored after Vedolizumab Treatment and May Predict Clinical Response in Inflammatory Bowel Disease Patients.

Vedolizumab (VDZ) is used for treating inflammatory bowel disease (IBD) patients. A study investigating colonic epithelial barrier function ex vivo following VDZ is lacking. This work aims to evaluate ex vivo the colonic epithelial barrier function in IBD patients at baseline and during VDZ treatment, and to investigate the relationships between barrier function and clinical parameters. Colonic specimens were obtained from 23 IBD patients before, and at 24 and 52 weeks after VDZ treatment, and from 26 healthy volunteers (HV). Transepithelial electrical resistance (TEER, permeability to ions) and paracellular permeability were measured in Ussing chambers. IBD patients showed increased epithelial permeability to ions (TEER, 13.80 ± 1.04 Ω × cm2 vs. HV 20.70 ± 1.52 Ω × cm2, p < 0.001) without changes in paracellular permeability of a 4 kDa probe. VDZ increased TEER (18.09 ± 1.44 Ω × cm2, p < 0.001) after 52 weeks. A clinical response was observed in 58% and 25% of patients at week 24, and in 62% and 50% at week 52, in ulcerative colitis and Crohn's disease, respectively. Clinical and endoscopic scores were strongly associated with TEER. TEER < 14.65 Ω × cm2 predicted response to VDZ (OR 11; CI 2-59). VDZ reduces the increased permeability to ions observed in the colonic epithelium of IBD patients before treatment, in parallel to a clinical, histological (inflammatory infiltrate), and endoscopic improvement. A low TEER predicts clinical response to VDZ therapy.

The role of claudins in homeostasis.

Sequential expression of claudins, a family of tight junction proteins, along the nephron mirrors the sequential expression of ion channels and transporters. Only by the interplay of transcellular and paracellular transport can the kidney efficiently maintain electrolyte and water homeostasis in an organism. Although channel and transporter defects have long been known to perturb homeostasis, the contribution of individual tight junction proteins has been less clear. Over the past two decades, the regulation and dysregulation of claudins have been intensively studied in the gastrointestinal tract. Claudin expression patterns have, for instance, been found to be affected in infection and inflammation, or in cancer. In the kidney, a deeper understanding of the causes as well as the effects of claudin expression alterations is only just emerging. Little is known about hormonal control of the paracellular pathway along the nephron, effects of cytokines on renal claudin expression or relevance of changes in paracellular permeability to the outcome in any of the major kidney diseases. By summarizing current findings on the role of specific claudins in maintaining electrolyte and water homeostasis, this Review aims to stimulate investigations on claudins as prognostic markers or as druggable targets in kidney disease.

NO Synthesis but Not Apoptosis, Mitosis or Inflammation Can Explain Correlations between Flow Directionality and Paracellular Permeability of Cultured Endothelium.

Haemodynamic wall shear stress varies from site to site within the arterial system and is thought to cause local variation in endothelial permeability to macromolecules. Our aim was to investigate mechanisms underlying the changes in paracellular permeability caused by different patterns of shear stress in long-term culture. We used the swirling well system and a substrate-binding tracer that permits visualisation of transport at the cellular level. Permeability increased in the centre of swirled wells, where flow is highly multidirectional, and decreased towards the edge, where flow is more uniaxial, compared to static controls. Overall, there was a reduction in permeability. There were also decreases in early- and late-stage apoptosis, proliferation and mitosis, and there were significant correlations between the first three and permeability when considering variation from the centre to the edge under flow. However, data from static controls did not fit the same relation, and a cell-by-cell analysis showed that <5% of uptake under shear was associated with each of these events. Nuclear translocation of NF-κB p65 increased and then decreased with the duration of applied shear, as did permeability, but the spatial correlation between them was not significant. Application of an NO synthase inhibitor abolished the overall decrease in permeability caused by chronic shear and the difference in permeability between the centre and the edge of the well. Hence, shear and paracellular permeability appear to be linked by NO synthesis and not by apoptosis, mitosis or inflammation. The effect was mediated by an increase in transport through tricellular junctions.

Brain microvascular endothelial cell dysfunction in an isogenic juvenile iPSC model of Huntington’s disease

Huntington’s disease (HD) is an inherited neurodegenerative disease caused by expansion of cytosine–adenine–guanine (CAG) repeats in the huntingtin gene, which leads to neuronal loss and decline in cognitive and motor function. Increasing evidence suggests that blood–brain barrier (BBB) dysfunction may contribute to progression of the disease. Studies in animal models, in vitro models, and post-mortem tissue find that disease progression is associated with increased microvascular density, altered cerebral blood flow, and loss of paracellular and transcellular barrier function. Here, we report on changes in BBB phenotype due to expansion of CAG repeats using an isogenic pair of induced pluripotent stem cells (iPSCs) differentiated into brain microvascular endothelial-like cells (iBMECs). We show that CAG expansion associated with juvenile HD alters the trajectory of iBMEC differentiation, producing cells with ~ two-fold lower percentage of adherent endothelial cells. CAG expansion is associated with diminished transendothelial electrical resistance and reduced tight junction protein expression, but no significant changes in paracellular permeability. While mutant huntingtin protein (mHTT) aggregates were not observed in HD iBMECs, widespread transcriptional dysregulation was observed in iBMECs compared to iPSCs. In addition, CAG expansion in iBMECs results in distinct responses to pathological and therapeutic perturbations including angiogenic factors, oxidative stress, and osmotic stress. In a tissue-engineered BBB model, iBMECs show subtle changes in phenotype, including differences in cell turnover and immune cell adhesion. Our results further support that CAG expansion in BMECs contributes to BBB dysfunction during HD.

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

Paracellular permeability changes induced by multi-walled carbon nanotubes in brain endothelial cells and associated roles of hemichannels

Multi-walled carbon nanotubes (MWCNTs) have promising applications in neurology depending on their unique physicochemical properties. However, there is limited understanding of their impacts on brain microvascular endothelial cells, the cells lining the vessels and maintaining the low and selective permeability of the blood-brain barrier. In this study, we examined the influence of pristine MWCNT (p-MWCNT) and carboxylated MWCNT (c-MWCNT) on permeability and tight junction tightness of murine brain microvascular endothelial cells, and investigated the potential mechanisms in the sight of hemichannel activity. Treatment with p-MWCNT for 24 h at subtoxic concentration (20 μg/mL) decreased the protein expression of occludin, disrupted zonula occludens-1 continuity, and elevated monolayer permeability as quantified by transendothelial electrical resistance and paracellular flux of 4000 Da fluorescein isothiocyanate-dextran conjugates. Moreover, p-MWCNT exposure also increased hemichannel activity with upregulated protein expression and altered subcellular localization of connexin (Cx)43 and pannexin (Panx)1. p-MWCNT-induced elevation in endothelial permeability could be prevented by hemichannel inhibitor carbenoxolone and peptide blocker of Cx43 and Panx1, indicating the crucial role of activated Cx43 and Panx1 hemichannels. Furthermore, Cx43 and Panx1 hemichannel-mediated ATP release might be involved in p-MWCNT-induced rise in endothelial permeability. In contrast, the above effects caused by p-MWCNT were not observed in cells treated with c-MWCNT, the functionalized form with more stable dispersion and a lower tendency to aggregate. Our study contributes further understanding of the impact of MWCNTs on brain endothelial tightness and permeability, which may have important implications for the safety application of MWCNTs in nanomedicine.

Unraveling the Pathology of the Blood‐Tumor Barrier in an Experimental Model of Brain Metastases of Lung Cancer

Lung cancer is the leading cause of cancer‐related deaths worldwide (1.76 million deaths in 2018) and the leading cause of brain metastatic disease among all primary cancers. As tumor cells colonize the neuroparenchyma, they breach the tightest and most effective vascular barrier in the body, the blood‐brain barrier (BBB). The functional components of the BBB include endothelial cells, basement membranes, pericytes and astrocytes. Herein, we hypothesized that dynamic transformation of the BBB to the blood‐tumor barrier (BTB) would correlate with changes in paracellular permeability. An experimental model of lung cancer brain metastases was developed using twelve 6‐week‐old athymic nude mice. Mice were injected with 250,000 brain‐seeking cells, which colonized the brain for 4–6 weeks. Animals were injected with 3 kd Texas Red dextran at the time of euthanasia, and brains were harvested, cryosectioned and prepared for immunofluorescence analysis. Metastatic tumors were characterized as highly or poorly permeable based on the diffusion of Texas Red dextran within and around the tumor parenchyma. Brain metastases were roughly spherical and measured between 50–500 μm in diameter, irrespective of their permeability status. The most striking BTB pathology was identified in tight junctions and associated adapter proteins. Highly permeable tumors exhibited haphazard expression of claudin‐5 and a loss of zona occludens‐1 adapter protein compared to metastases with low paracellular permeability. Understanding the cellular and molecular alterations occurring within the BBB during brain metastatic disease from non‐small cell lung cancer is imperative for the identification of novel therapeutic targets and improved methods of drug delivery.Support or Funding InformationClaudine Auld was supported by the Summer Research Opportunities Program, sponsored by the American Society of Investigative Pathology. This research was supported in part by the Showalter Research Trust.

Paracellular transport to the coral calcifying medium: effects of environmental parameters.

Coral calcification relies on the transport of ions and molecules to the extracellular calcifying medium (ECM). Little is known about paracellular transport (via intercellular junctions) in corals and other marine calcifiers. Here, we investigated whether the permeability of the paracellular pathway varied in different environmental conditions in the coral Stylophora pistillata Using the fluorescent dye calcein, we characterised the dynamics of calcein influx from seawater to the ECM and showed that increases in paracellular permeability (leakiness) induced by hyperosmotic treatment could be detected by changes in calcein influx rates. We then used the calcein-imaging approach to investigate the effects of two environmental stressors on paracellular permeability: seawater acidification and temperature change. Under conditions of seawater acidification (pH 7.2) known to depress pH in the ECM and the calcifying cells of S. pistillata, we observed a decrease in half-times of calcein influx, indicating increased paracellular permeability. By contrast, high temperature (31°C) had no effect, whereas low temperature (20°C) caused decreases in paracellular permeability. Overall, our study establishes an approach to conduct further in vivo investigation of paracellular transport and suggests that changes in paracellular permeability could form an uncharacterised aspect of the physiological response of S. pistillata to seawater acidification.

Recombinant &lt;i&gt;L. lactis&lt;/i&gt; Expressing Defensin-5 Protects Against Mice Colitis via NF-κβ Pathway

Defensin is a key antimicrobial peptide which plays an important role in host immunity and maintaining gut barrier function. In this study, we optimized a defensin mHD-5 clone primarily consisting of the mature human defensin-5 (HD-5) peptide, constructed the pN8148-SHD-5 vector, and transfected this plasmid into Lactococcus lactis to create the recombinant NZ9000SHD-5 strain, which produces HD-5 under nisin-controlled gene expression. We found exposure of mice with colitis induced by dextran sodium sulfate (DSS) to the NZ9000SHD-5 strain attenuated inflammatory cell infiltration and histopathological changes in colonic glands and preserved epithelial barrier integrity. The same protective effects were observed in RAW264.7 cells and Caco-2 cells in in vitro experiments. Moreover, pretreatment of the mouse macrophage cell line RAW264.7 with NZ9000SHD-5 culture supernatants prior to LPS application significantly reduced the expression of phosphorylated nuclear transcription factor-kappa B (NF-κβ) p65 and its inhibitor IκBα. These results indicate the NZ9000SHD-5 strain can alleviate DSS-induced mucosal damage and changes in paracellular permeability, possibly by attenuating colonic inflammation through downregulation of the NF-κβ signaling pathway. Thus, administration of the NZ9000SHD-5 strain may be a possible therapeutic option for colitis. Funding: Supported by the National Natural Science Foundation of China (No. 81570480 and 81770529) and the Special Scientific Research Fund of Public Welfare Profession of National Health and Family Planning Commission (No. 201502026). Declaration of Interest: The author have claimed that there was no competing interest. Ethical Approval: The Southern Medical University Ethics Committee approved the protocols according to the Helsinki Declaration (6th revision, 2008), and all animal procedures were approved by the Institutional Animal Care and Use Committee of Southern Medical University.

Activation of calcium-sensing receptor by allosteric agonists cinacalcet and AC-265347 abolishes the 1,25(OH)2D3-induced Ca2+ transport: Evidence that explains how the intestine prevents excessive Ca2+ absorption

Long-term high-calcium intake and intestinal calcium hyperabsorption are hazardous to the body. It is hypothesized that enterocytes possess mechanisms for preventing superfluous calcium absorption, including secretion of negative regulators of calcium absorption and utilization of calcium-sensing receptor (CaSR) to detect luminal calcium. Herein, Caco-2 monolayers were treated with high doses of 1,25(OH)2D3 to induce calcium hyperabsorption or directly exposed to high apical calcium. The expression of counterregulatory factor of calcium absorption, fibroblast growth factor (FGF)-23, was also investigated in the intestine of lactating rats, which physiologically exhibit calcium hyperabsorption. We found that FGF-23 expression was enhanced in all intestinal segments of lactating rats. In Caco-2 monolayers, high apical calcium and 1,25(OH)2D3 induced FGF-23 secretion into culture media. FGF-23 antagonized 1,25(OH)2D3-induced calcium transport and led to a significant, but small, change in paracellular permeability. Furthermore, high-dose 1,25(OH)2D3 upregulated FGF-23 expression, which was prevented by CaSR inhibitors. Activation of apical CaSR by cinacalcet and AC-265347 abolished 1,25(OH)2D3-induced calcium transport in a dose-dependent manner. In conclusion, the intestinal FGF-23 expression was upregulated in conditions with calcium hyperabsorption, presumably to help protect against excessive calcium absorption, while CaSR probably monitored calcium in the lumen and induced FGF-23 production for preventing superfluous calcium uptake.

Effect of glycated insulin on the blood-brain barrier permeability: An in vitro study

Altered blood-brain barrier (BBB) permeability may contribute to pathogenesis of diabetes-related central nervous system disorders. Considering the presence of glycated insulin in plasma of type 2 diabetic patients, we hypothesized that glycated insulin could induce changes in paracellular permeability in BBB. Therefore, the authors decided to study the effect of glycated insulin on paracellular permeability in a BBB model and the change induced in insulin conformation upon glycation.In this study, the structural modification was examined by fluorescence and circular dichroism spectroscopies and dynamic light scattering. Cell proliferation and production of ROS in astrocytes and HUVEC cells were analyzed by MTT and spectrofluorometric assays, respectively. Apoptosis induction was determined and confirmed by flow cytometry and western blot analyses, respectively. The permeability was measured Lucifer yellow and FITC-Dextran.According to our results, glycated insulin presented altered conformation and more exposed hydrophobic patches than insulin. Formation of oligomeric species and advanced glycated end products (AGEs) were determined. Lower cell viability, higher apoptosis, and more ROS were detected upon treatment of cells with glycated insulin. Finally, glycated insulin led to increased Lucifer yellow and FITC-dextran transportation across the BBB model which could result from ROS producing and apoptosis-inducing activities of AGE-insulin.

Changes in paracellular permeability induced by Pepsin-Trypsin digested Gliadin (PTG): Role of polyamines in the Lactobacillus rhamnosus GG protective action

Celiac disease (CD) is an immune-mediated enteropathy characterized by an increase in paracellular permeability. The improvements in barrier integrity have been related to changes in tight junction (TJ) structure as a consequence of modifications in TJ protein expression. Recently, also polyamines have been suggested to play a part in the control of intestinal permeability by modulating TJ and adherens junction (AJ) expressions.The present work investigated the protective role of Lactobacillus rhamnosus GG (L.GG) towards alterations of the paracellular permeability induced by Pepsin-Trypsin digested Gliadin (PTG) in Caco-2 cells.The results from this study clearly indicate that L.GG protects Caco-2 cells from PTG induced damage and, interestingly, the presence of cellular polyamines seems to be essential in the modulation of TJ and AJ protein expression exerted by this probiotic. On these bases, a role for L.GG as a dietary supplement to promote health also for CD patients could be hypothesized.

Th17 Cytokines Disrupt the Airway Mucosal Barrier in Chronic Rhinosinusitis.

Cytokine mediated changes in paracellular permeability contribute to a multitude of pathological conditions including chronic rhinosinusitis (CRS). The purpose of this study was to investigate the effect of interferons and of Th1, Th2, and Th17 cytokines on respiratory epithelium barrier function. Cytokines and interferons were applied to the basolateral side of air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) from CRS with nasal polyp patients. Transepithelial electrical resistance (TEER) and permeability of FITC-conjugated dextrans were measured over time. Additionally, the expression of the tight junction protein Zona Occludens-1 (ZO-1) was examined via immunofluorescence. Data was analysed using ANOVA, followed by Tukey HSD post hoc test. Our results showed that application of interferons and of Th1 or Th2 cytokines did not affect the mucosal barrier function. In contrast, the Th17 cytokines IL-17, IL-22, and IL-26 showed a significant disruption of the epithelial barrier, evidenced by a loss of TEER, increased paracellular permeability of FITC-dextrans, and discontinuous ZO-1 immunolocalisation. These results indicate that Th17 cytokines may contribute to the development of CRSwNP by promoting a leaky mucosal barrier.

VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

Neurological disorders are associated with an increase in the permeability of human brain microvascular endothelial cells (HBMEC). Our previous findings have indicated that EphA2 could increase the permeability of HBMEC. Recent evidence has linked EphA2 and vascular endothelial growth factor (VEGF) to abnormalities in the vascular response. However, it is unclear whether EphA2 is involved in the VEGF-induced changes in the permeability of HBMEC. Here, changes in permeability were determined by measuring transendothelial electrical resistance (TEER) and the flux of FITC-dextran. We found that knockdown of EphA2 in HBMEC abolished the VEGF-induced reduction in TEER and increase in flux of fluorescent dextran. Moreover, VEGF-induced redistribution of ZO-1 and the recruitment of detergent-soluble occludin and claudin-5 were also prevented. Further results showed that VEGF increased EphA2 expression in a time- and dose-dependent manner, which was inhibited by a neutralizing antibody against VEGFR2 or SU1498. VEGF-induced EphA2 expression was suppressed in the brain endothelium following treatments with the PI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant-negative PI3K mutants (Δp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Our data suggest that VEGF upregulates the expression of EphA2 in HBMEC through binding to VEGFR2 and subsequently activating the intracellular PI3K/Akt and ERK1/2 signaling pathways, which contribute to an increase in paracellular permeability. These data reveal a novel role for VEGF as a regulator of EphA2 expression in the brain endothelial cells and provide insights into the molecular mechanisms of VEGF-mediated changes in paracellular permeability.

Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response.

Triptolide alters barrier function in renal proximal tubular cells in rats

Alteration of the tight junction complex in renal epithelial cells can affect renal barrier function and perturb normal kidney homeostasis. The objective of the present study was to determine whether triptolide could affect tight junctions in the proximal tubule epithelial cells both in vivo and in vitro. Wistar rats were gavaged with triptolide at 0, 100, 200 or 400μg/kg/day for 28 days. Pathologic examination of the kidney showed that triptolide primarily affected the proximal tubules. The nephrotoxicity of triptolide is morphologically characterized by the detachment of the proximal tubular epithelial cells from each other. Immunohistochemical analysis showed that there was marked alteration in the localization of Zonula Occludens 1 protein (ZO-1) in the proximal tubule epithelium. Additionally, the uptake of FITC-dextran, a marker of fluid phase endocytosis in the proximal tubule, was considerably lower in triptolide-treated animals than in normal rats. Supported by these results, we detected significant increases in blood urea nitrogen (BUN) but not of creatinine (Cr) in rats treated with triptolide, indicating damage to the proximal tubules. Furthermore, triptolide treatment caused an alteration of the tight junction complex, resulting in changes in paracellular permeability in NRK-52E cells in vitro. Taken together, these results suggest that triptolide induced renal toxicity in rats and that the mechanism of toxicity was related to the disruption of cell–cell junctions and alterations of the paracellular permeability in the proximal tubule.

TNF-α–mediated bronchial barrier disruption and regulation by src-family kinase activation

Because TNF-α is increased in severe asthma, we hypothesized that TNF-α contributes to barrier dysfunction and cell activation in bronchial epithelial cells. We further hypothesized that src-family kinase inhibition would improve barrier function in healthy cells in the presence of TNF-α and directly in cultures of severe asthmatic cells where the barrier is disrupted. We assessed the effect of TNF-α, with or without src-family kinase inhibitor SU6656, on barrier properties and cytokine release in differentiated human bronchial epithelial cultures. Further, we tested the effect of SU6656 on differentiated primary cultures from severe asthma. Barrier properties of differentiated human bronchial epithelial air-liquid interface cultures from healthy subjects and subjects with severe asthma were assessed with transepithelial electrical resistance and fluorescent dextran passage. Proteins were detected by immunostaining or Western blot analysis and cytokines by immunoassay. Mechanisms were investigated with src kinase and other inhibitors. TNF-α lowered transepithelial electrical resistance and increased fluorescent dextran permeability, caused loss of occludin and claudins from tight junctions with redistribution of p120 catenin and E-cadherin from adherens junctions, and also increased endogenous TNF-α, IL-6, IL-1β, IL-8, thymic stromal lymphoprotein, and pro-matrix metalloprotease 9 release. SU6656 reduced TNF-α-mediated paracellular permeability changes, restored occludin, p120, and E-cadherin and lowered autocrine TNF-α release. Importantly, SU6656 improved the barrier properties of severe asthmatic air-liquid interface cultures. Redistribution of E-cadherin and p120 was observed in bronchial biopsies from severe asthmatic airways. Inhibiting TNF-α or src kinases may be a therapeutic option to normalize barrier integrity and cytokine release in airway diseases associated with barrier dysfunction.

Translational Approaches for Pharmacotherapy Development for Acute Diarrhea

Areport is presented of a National Institute of Diabetes and Digestive and Kidney Diseases–sponsored single topic conference designed to determine scientific advances needed to encourage development of pharmacotherapy for diarrheal diseases. Acute diarrheal diseases are a global public health problem in developing and developed countries. They are a leading cause of mortality and morbidity for children under the age of 5 worldwide and have a major economic impact in developed countries. Although oral rehydration solution (ORS) along with oral zinc therapy has reduced mortality owing to acute diarrheal diseases, there are essentially no other approved, safe, and effective drugs to decrease stool volume and prevent fluid loss. On September 25–27, 2011, the National Institute of Diabetes and Digestive and Kidney Diseases convened a workshop on Translational Approaches for Pharmacotherapy Development for Acute Diarrhea to assess current treatments for acute diarrhea, to identify common host pathways targeted by infectious agents associated with acute diarrhea, to evaluate which pathophysiologic mechanisms are most clinically relevant as potential targets, and to identify promising areas for translational research.

Manipulation of Cell Physiology Enables Gene Silencing in Well-differentiated Airway Epithelia

The application of RNA interference-based gene silencing to the airway surface epithelium holds great promise to manipulate host and pathogen gene expression for therapeutic purposes. However, well-differentiated airway epithelia display significant barriers to double-stranded small-interfering RNA (siRNA) delivery despite testing varied classes of nonviral reagents. In well-differentiated primary pig airway epithelia (PAE) or human airway epithelia (HAE) grown at the air–liquid interface (ALI), the delivery of a Dicer-substrate small-interfering RNA (DsiRNA) duplex against hypoxanthine–guanine phosphoribosyltransferase (HPRT) with several nonviral reagents showed minimal uptake and no knockdown of the target. In contrast, poorly differentiated cells (2–5-day post-seeding) exhibited significant oligonucleotide internalization and target knockdown. This finding suggested that during differentiation, the barrier properties of the epithelium are modified to an extent that impedes oligonucleotide uptake. We used two methods to overcome this inefficiency. First, we tested the impact of epidermal growth factor (EGF), a known enhancer of macropinocytosis. Treatment of the cells with EGF improved oligonucleotide uptake resulting in significant but modest levels of target knockdown. Secondly, we used the connectivity map (Cmap) database to correlate gene expression changes during small molecule treatments on various cells types with genes that change upon mucociliary differentiation. Several different drug classes were identified from this correlative assessment. Well-differentiated epithelia treated with DsiRNAs and LY294002, a PI3K inhibitor, significantly improved gene silencing and concomitantly reduced target protein levels. These novel findings reveal that well-differentiated airway epithelia, normally resistant to siRNA delivery, can be pretreated with small molecules to improve uptake of synthetic oligonucleotide and RNA interference (RNAi) responses.