Epithelial Tight Junction Proteins Research Articles

Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner

Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as nonbloody diarrhea may be Stx independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria with the cecum and colon. Greater numbers of EHEC were also cultured from stool samples obtained from streptomycin-pretreated mice, as compared with those that received no antibiotics. Transmission electron microscopy shows that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of the colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in the colonic tissues of infected animals. Increased mucosal permeability to 4- kDa FITC-dextran was also observed in the colonic tissues of infected mice. Immunofluorescence microscopy showed that EHEC infection resulted in redistribution of the tight junction (TJ) proteins occludin and claudin-3 and increased the expression of claudin-2, whereas ZO-1 localization remained unaltered. Quantitative real-time PCR showed that EHEC altered mRNA transcription of OCLN, CLDN2, and CLDN3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of TJ proteins has a role in the increase in intestinal permeability.

Effects of Bawei Xilei San on mice with oxazolone-induced colitis and the mechanisms

To investigate the therapeutic effects of Bawei Xilei San (BWXLS), a compound traditional Chinese herbal medicine, on mice with oxazolone-induced colitis and to explore the mechanisms. Thirty-two BALB/c mice were randomly divided into 4 groups (8 for each): normal control group, untreated group, hydrocortisone group and BWXLS group. Except for the mice in the normal control group, all mice were intrarectally administered with 3.0% oxazolone to induce colitis. Then the mice in the normal control group and untreated group were administered with 0.9% carboxymethyl cellulose sodium solution. Mice in the BWXLS group were intrarectally administered with 0.2 mg/g BWXLS and hydrocortisone group with 0.02 mg/g respectively for 5 days. The body weight and stool consistency and occult or gross blood were recorded to calculate the disease activity index (DAI). The mice were sacrificed at the 6th day. The macroscopic and histological changes of the colon were evaluated. The expressions of Toll-like receptor 4 (TLR4), nuclear factor-kappaB (NF-kappaB), and epithelial tight junction protein occludin were assessed by immunohistochemical method. The level of tumor necrosis factor-alpha (TNF-alpha) in colonic mucosa was evaluated by enzyme-linked immunosorbent assay. The DAI, and macroscopic and histological changes in the BWXLS group were improved as compared with those in the untreated group (P<0.05) but were similar to those in the hydrocortisone group. The expression of occludin was significantly increased (P<0.05) while the expressions of TLR4, NF-kappaB and TNF-alpha were significantly decreased in the BWXLS group as compared with the untreated group, and were similar to those in the hydrocortisone group (P>0.05). Up-regulating the expression of occludin and down-regulating the expressions of TLR4 and NF-kappaB, and hence inhibiting TNF-alpha expression and improving the mucosa barrier function may be part of the mechanisms of BWXLS in treating oxazolone-induced colitis in mice.

Elevated cysteine-protease activity in fecal supernatants of constipation predominant IBS patients: a new factor disrupting colonic epithelial barrier

Fecal serine-protease activity is elevated in diarrhea type irritable bowel syndrome (IBS-D), and this luminal factor enhances colonic permeability and colorectal sensitivity in mice when infused intracolonically (ic.). Rectal hypersensitivity has been described in all IBS subgroups, including constipation predominant IBS (IBS-C). Other proteases, such as cysteine-proteases (CP) are present in feces, and these proteases were found capable of digesting epithelial tight junction proteins in the human airways. Aims: To compare fecal CP levels in IBS-C and IBS-D patients (pts) with healthy controls, and to test the effect of IBS-C supernatants on colorectal sensitivity of mice. Methods: Fecal samples were collected from healthy subjects (n=20), pts with IBS-C (n=25) and IBS-D (n=26). CP activity was determined by a fluorometric assay. Visceral hypersensitivity was assessed in C57/BL6 mice by recording abdominal contractions provoked by colorectal distension (CRD). 4 days after insertion of electrodes in the abdominal muscle, mice received ic. 0.3ml of fecal supernatants from IBS-C pts or controls 2 times with 12h interval. CRD was performed 1h after the end of infusion. A group of mice received IBS-C supernatants previously incubated with the specific inhibitor of CP (E64). Results: Fecal CP activity is markedly elevated in IBS-C pts compared to controls (2420±718 vs. 399±190 Δfluo/mg prot, p&lt;0.05). In contrast, there is no significant elevation in the level of CP activity in IBS-D. Ic. administration of IBS-C supernatants increased the abdominal EMG response to distension by 377, 164, 53 and 20% at distension volumes from 0.02 to 0.08 mL, respectively (p&lt;0.05). The inhibition of CP activity blocked the pronociceptive effect completely. Conclusions: Fecal CP activity is elevated in IBS-C. Fecal supernatants from IBS-C pts provoke visceral hypersensitivity to distension in mice, which effect is specific to CP. This may be a new factor that could contribute to the genesis of symptoms in IBS-C. (Grant: TÁMOP-4.2.2–08)

Regulation of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective effects on the epithelial barrier

Lactobacillus plantarum, a commensal bacterium of humans, has been proposed to enhance the intestinal barrier, which is compromised in a number of intestinal disorders. To study the effect of L. plantarum strain WCFS1 on human barrier function, healthy subjects were administered L. plantarum or placebo in the duodenum for 6 h by means of a feeding catheter. The scaffold protein zonula occludens (ZO)-1 and transmembrane protein occludin were found to be significantly increased in the vicinity of the tight-junction (TJ) structures, which form the paracellular seal between cells of the epithelium. In an in vitro model of the human epithelium, L. plantarum induced translocation of ZO-1 to the TJ region; however, the effects on occludin were minor compared with those seen in vivo. L. plantarum was shown to activate Toll-like receptor 2 (TLR2) signaling, and treatment of Caco-2 monolayers with the TLR2 agonist Pam(3)-Cys-SK4(PCSK) significantly increased fluorescent staining of occludin in the TJ. Pretreatment of Caco-2 monolayers with L. plantarum or PCSK significantly attenuated the effects of phorbol ester-induced dislocation of ZO-1 and occludin and the associated increase in epithelial permeability. Our results identifying commensal bacterial stimulation of TLR2 in the gut epithelium as a regulator of epithelial integrity have important implications for understanding probiotic mechanisms and the control of intestinal homeostasis.

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.

Probiotic mixture VSL#3 protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in a murine model of colitis

Changes in epithelial tight junction protein expression and apoptosis increase epithelial permeability in inflammatory bowel diseases. The effect of the probiotic mixture VSL#3 on the epithelial barrier was studied in dextran sodium sulfate (DSS)-induced colitis in mice. Acute colitis was induced in BALB/c mice (3.5% DSS for 7 days). Mice were treated with either 15 mg VSL#3 or placebo via gastric tube once daily during induction of colitis. Inflammation was assessed by clinical and histological scores. Colonic permeability to Evans blue was measured in vivo. Tight junction protein expression and epithelial apoptotic ratio were studied by immunofluorescence and Western blot. VSL#3 treatment reduced inflammation (histological colitis scores: healthy control 0.94 +/- 0.28, DSS + placebo 14.64 +/- 2.55, DSS + VSL#3 8.43 +/- 1.82; P = 0.011). A pronounced increase in epithelial permeability in acute colitis was completely prevented by VSL#3 therapy [healthy control 0.4 +/- 0.07 (extinction/g), DSS + placebo 5.75 +/- 1.67, DSS + VSL#3 0.26 +/- 0.08; P = 0.003]. In acute colitis, decreased expression and redistribution of the tight junction proteins occludin, zonula occludens-1, and claudin-1, -3, -4, and -5 were observed, whereas VSL#3 therapy prevented these changes. VSL#3 completely prevented the increase of epithelial apoptotic ratio in acute colitis [healthy control 1.58 +/- 0.01 (apoptotic cells/1,000 epithelial cells), DSS + placebo 13.33 +/- 1.29, DSS + VSL#3 1.72 +/- 0.1; P = 0.012]. Probiotic therapy protects the epithelial barrier in acute colitis by preventing 1) decreased tight junction protein expression and 2) increased apoptotic ratio.

HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction

BackgroundHIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined.ResultsHIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization.ConclusionTaken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.

Escherichia albertii and Hafnia alvei are candidate enteric pathogens with divergent effects on intercellular tight junctions

Attaching-effacing lesion-inducing Escherichia albertii and the related, but non-attaching–effacing organism, Hafnia alvei, are both implicated as enteric pathogens in humans. However, effects of these bacteria on epithelial cells are not well-characterized. Related enteropathogens, including enterohemorrhagic Escherichia coli O157:H7, decrease epithelial barrier function by disrupting intercellular tight junctions in polarized epithelia. Therefore, this study assessed epithelial barrier function and tight junction protein distribution in polarized epithelia following bacterial infections. Polarized epithelial (MDCK-I and T84) cells grown on filter supports were infected apically with E. coli O157:H7, E. albertii, and H. alvei for 16 h at 37 °C. All strains decreased transepithelial electrical resistance and increased permeability to a dextran probe in a host cell-dependent manner. Immunofluorescence microscopy showed that both E. coli O157:H7 and E. albertii, but not H. alvei, caused a redistribution of the tight junction protein zona occludens-1. In contrast to E. coli O157:H7, E. albertii and H. alvei did not redistribute claudin-1. Western blotting of whole cell protein extracts demonstrated that each bacterium caused differential changes in tight junction protein expression, dependent on the host cell. These findings demonstrate that E. albertii and H. alvei are candidate enteric pathogens that have both strain-specific and host epithelial cell-dependent effects.

Claudin-1 and claudin-2 expression is elevated in inflammatory bowel disease and may contribute to early neoplastic transformation

Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal adenocarcinoma. The factors that result in IBD-associated carcinogenesis are not understood. We hypothesized that altered expression of intestinal epithelial tight junction proteins might contribute to neoplastic progression. Semiquantitative immunohistochemical staining of human biopsies was used to assess expression of the tight junction proteins claudin-1, claudin-2, claudin-4, and occludin in IBD, IBD-associated dysplasia, acute, self-limited colitis (ASLC), and sporadic adenomas. Claudin-1 and claudin-2 expression was elevated in active IBD, adenomas, and IBD-associated dysplasia, but not ASLC. In contrast, claudin-4 expression was elevated in both active IBD and ASLC. Occludin expression was similar to control in all cases. Importantly, in IBD, claudin-1 and claudin-2 expression correlated positively with inflammatory activity. To investigate mechanisms underlying altered claudin expression, β-catenin activation was assessed as nuclear localization. Like claudin-1 and claudin-2, β-catenin was markedly activated in IBD, sporadic dysplasia, and IBD-associated dysplasia, but was only slightly activated in ASLC. Taken together, these data suggest that β-catenin transcriptional activity is elevated in chronic injury and that this may contribute to increased claudin-1 and claudin-2 expression. We speculate that increased claudin-1 and claudin-2 expression may be involved at early stages of transformation in IBD-associated neoplasia.

Secreted bioactive factors fromBifidobacterium infantisenhance epithelial cell barrier function

Live probiotic bacteria are effective in reducing gut permeability and inflammation. We have previously shown that probiotics release peptide bioactive factors that modulate epithelial resistance in vitro. The objectives of this study were to determine the impact of factors released from Bifidobacteria infantis on intestinal epithelial cell permeability and tight junction proteins and to assess whether these factors retain their bioactivity when administered to IL-10-deficient mice. B. infantis conditioned medium (BiCM) was applied to T84 human epithelial cells in the presence and absence of TNF-alpha and IFN-gamma. Transepithelial resistance (TER), tight junction proteins [claudins 1, 2, 3, and 4, zonula occludens (ZO)-1, and occludin] and MAP kinase activity (p38 and ERK) were examined. Acute effects of BiCM on intestinal permeability were assessed in colons from IL-10-deficient mice in Ussing chambers. A separate group of IL-1-deficient mice was treated with BiCM for 4 wk and then assessed for intestinal histological injury, cytokine levels, epithelial permeability, and immune response to bacterial antigens. In T84 cells, BiCM increased TER, decreased claudin-2, and increased ZO-1 and occludin expression. This was associated with enhanced levels of phospho-ERK and decreased levels of phospho-p38. BiCM prevented TNF-alpha- and IFN-gamma-induced drops in TER and rearrangement of tight junction proteins. Inhibition of ERK prevented the BiCM-induced increase in TER and attenuated the protection from TNF-alpha and IFN-gamma. Oral BiCM administration acutely reduced colonic permeability in mice whereas long-term BiCM treatment in IL-10-deficient mice attenuated inflammation, normalized colonic permeability, and decreased colonic and splenic IFN-gamma secretion. In conclusion, peptide bioactive factors from B. infantis retain their biological activity in vivo and are effective in normalizing gut permeability and improving disease in an animal model of colitis. The effects of BiCM are mediated in part by changes in MAP kinases and tight junction proteins.

Unique Role of Junctional Adhesion Molecule-A in Maintaining Mucosal Homeostasis in Inflammatory Bowel Disease

Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Control, Crohn's disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A(-/-), Tie-2-Cre-JAM-A(-/-) (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre-JAM-A(-/-) mice are no more susceptible to DSS colitis than wild-type animals. JAM-A(-/-) mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function.

Heparin blocks the adhesion of E. coli O157:H7 to human colonic epithelial cells

Adhesion of Shiga toxin-producing Enterohemorrhagic Escherichia coli (EHEC) O157:H7 to human colonic epithelium is a critical step for infection by this type of bacteria. Here, we demonstrate that adherence of EHEC O157:H7 to cultured human colonic T84 epithelial monolayers can be blocked by heparin and heparan sulfate in a dose-dependent fashion. In doing this, heparin and heparan sulfate also prevent dysfunction of the T84 barrier and disorganization of epithelial tight junction protein ZO-1 caused by EHEC O157:H7. This inhibition by heparin and heparan sulfate seems to result from a block in the binding interactions of bacteria intimin with epithelial β 1 integrins. This study provides evidence, for the first time, that heparin and heparan sulfate can serve as novel effective blockers in preventing EHEC O157:H7 infection.

Altered permeability in inflammatory bowel disease: pathophysiology and clinical implications

To present the mechanisms behind barrier disturbance in inflammatory bowel disease and their functional consequences. A reduction in tight junction strands, strand breaks and alteration of tight junction protein content and composition characterize Crohn's disease. In ulcerative colitis, epithelial leaks appear early as a result of microerosions, upregulated epithelial apoptosis and tight junction protein changes with pronounced increases in claudin-2. T-helper type 1 cytokine effects by interferon-gamma and tumour necrosis factor alpha are important for epithelial damage in Crohn's disease. Interleukin-13 is the key effector cytokine in ulcerative colitis, stimulating epithelial cell apoptosis, and can upregulate claudin-2 expression. Together with interleukin-13-induced epithelial restitution arrest, this may explain why ulcer lesions occur in early stages of ulcerative colitis but are only observed in advanced inflammatory stages in Crohn's disease. Barrier dysfunction in inflammatory bowel disease contributes to diarrhea by a leak flux mechanism and can cause mucosal inflammation secondary to luminal antigen uptake. Barrier abnormalities, such as epithelial tight junction changes and apoptotic leaks, gross mucosal lesions, and epithelial restitution arrest are responsible for these abnormalities and are the result of immune dysregulation. Studying the underlying mechanisms is important in understanding the pathophysiology of inflammatory bowel disease and developing therapeutic strategies.

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

Down-regulated expression of intestinal epithelial tight junction protein occludin in mice with fulminant hepatic failure

Down-regulated expression of intestinal epithelial tight junction protein occludin in mice with fulminant hepatic failure

Transforming Growth Factor-β Regulation of Epithelial Tight Junction Proteins Enhances Barrier Function and Blocks Enterohemorrhagic Escherichia coli O157:H7-Induced Increased Permeability

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an enteric pathogen that causes potentially fatal symptoms after intimate adhesion, modulation of intestinal epithelial signal transduction, and alteration of epithelial function (eg, barrier disruption). Although the epithelial barrier is critical to gut homeostasis, only a few agents, such as transforming growth factor (TGF)-beta, can enhance or protect epithelial barrier function. Our aims were to delineate the mechanism(s) behind TGF-beta-induced barrier enhancement and to determine whether TGF-beta could prevent EHEC-induced barrier disruption. Using monolayers of the human T84 colonic epithelial cell line, we found that TGF-beta induced a significant increase in transepithelial electrical resistance (a measure of paracellular permeability) through activation of ERK MAPK and SMAD signaling pathways and up-regulation of the tight junction protein claudin-1. Additionally, TGF-beta pretreatment of epithelia blocked the decrease in transepithelial electrical resistance and the increase in transepithelial passage of [(3)H]-mannitol caused by EHEC infection. EHEC infection was associated with reduced expression of zonula occludens-1, occludin, and claudin-2 (but not claudin-1 or claudin-4); TGF-beta pretreatment prevented these changes. These studies provide insight into EHEC pathogenesis by illustrating the mechanisms underlying TGF-beta-induced epithelial barrier enhancement and identifying TGF-beta as an agent capable of blocking EHEC-induced increases in epithelial permeability via maintenance of claudin-2, occludin, and zonula occludens-1 levels.

Mechanism of IFN-γ-induced Endocytosis of Tight Junction Proteins: Myosin II-dependent Vacuolarization of the Apical Plasma Membrane

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.

Interferon‐γ induces internalization of epithelial tight junction proteins via a macropinocytosis‐like process

Increased epithelial permeability is observed in inflammatory states. However, the mechanism by which inflammatory mediators such as IFN-gamma increase epithelial permeability is unknown. We recently observed that IFN-gamma induces disassembly of tight junctions (TJ); in this study we asked whether such TJ disassembly is mediated by endocytosis of junctional proteins. The role of three major internalization pathways in disruption of TJ in IFN-gamma-treated intestinal epithelial cells was analyzed using selective inhibitors and markers of the pathways. No role for the clathrin- and caveolar-mediated endocytosis in the IFN-gamma-induced internalization of TJ proteins was observed. However, inhibitors of macropinocytosis blocked internalization of TJ proteins and junctional proteins colocalized with macropinocytosis markers, dextran and phosphatidylinositol-3,4,5-trisphosphate. Internalized TJ proteins were identified in early and recycling endosomes but not in late endosomes/lysosomes. These results for the first time suggest that IFN-gamma produces a leaky epithelial barrier by inducing macropinoytosis of TJ proteins.

Epithelial tight junction proteins as potential antibody targets for pancarcinoma therapy.

Recombinant monoclonal antibodies are beginning to revolutionize cancer therapy. In combination with standard chemotherapy, high response rates have been reported with antibodies of the human IgG1 isotype for treatment of non-Hodgkin's lymphoma and breast cancer. It is becoming apparent that targets for antibody-based therapies do not necessarily need to be absent from normal tissues but can be present there either in low copy numbers or with binding epitopes shielded from the therapeutic antibody. Here, we studied whether claudin proteins that form tight junctions in normal epithelia are still expressed on carcinoma cells and whether their extracellular domains can be recognized by antibodies. We show that mRNAs of claudins 1, 3, 4, and 7 are all expressed in different human carcinoma cell lines, while claudin 8 was selectively expressed in breast and pancreas cancer lines. Chicken polyclonal antibodies were raised against peptides contained within predicted extracellular domains of claudins 1, 3, and 4. Affinity-purified IgG fractions for claudins 3 and 4 were monospecific and bound to human breast and colon carcinoma lines, but not to a line of monocytic origin. Claudin 3 antibodies also homogeneously stained human renal cell carcinoma tissue and micrometastatic tumor cells as identified by cytokeratin staining in bone marrow biopsies of breast cancer patients. Fluorescence-activated cell sorting and immunocytochemistry indicated that claudin antibodies bound to the surface of tumor cells. By analogy to other tumor-associated antigens that are differentially accessible to antibodies on tumor vs normal tissue, we propose that certain claudin proteins have potential as targets for novel antibody-based therapies of carcinomas.

514. Potential of Bone Marrow Mesenchymal Stem Cells for Cystic Fibrosis Therapy

Bone marrow mesenchymal stem cells (MSCs) have been shown to differentiate into pulmonary epithelial cells in animal models. To explore whether human MSCs can undergo differentiation into airway epithelia, which can be exploited as a novel gene therapy strategy for cystic fibrosis (CF), human MSCs expressing green florescent protein (GFP) were mixed with primary human airway epithelial cells in a ratio of 1:5, 1:10, or 1:20, and seeded on a semi-permeable filter membrane coated with human placenta collagen type VI. The cells were cultured at an air-liquid interface and were able to maintain the apical surface dry after one to two weeks with transepithelial resistance greater than 700 Ohms, suggesting the formation of tight junctions. Immunofluorescent staining with an antibody against cytokeratin 18 (CK18) demonstrated that the stem cells co-cultured with human airway cells expressed this epithelium-specific marker, whereas prior to the culture, there is no detectable CK18 expression by this approach. Morphologically, the GFP-labeled stem cells converted their original thin-spindle fibroblast shape or flat polygonal cell shape to a typical epithelial cuboidal shape with an accessory structure like cilia by confocal microscopy, suggesting that the hMSCs in the co-culture system transdifferentiate into airway epithelial cells. To confirm the finding, we further did immunostaining with an antibody against occludin, an epithelial tight junction protein. Flow cytometry and fluorescent microscopy showed that ~10% of the initially loaded GFP-MSCs express this critical epithelial marker. By cell sorting, we isolated the co-cultured hMSCs, and RT-PCR was performed with primers specific to human CFTR. The results demonstrated that the air-liquid culture induces hMSCs to express the CFTR gene. Furthermore, co-culture of normal hMSCs with CF airway epithelial cells resulted in the wild-type CFTR expression. More importantly, the MSCs isolated from CF patients were able to be gene-corrected. In vitro differentiation assays proved that the CFTR gene-corrected CF MSCs still possess their naive multipotency, such as differentiation along osteogenic, adipogenic, and chondrogenic pathways. The gene-corrected CF MSCs were able to contribute to apical Cl- secretion by chloride efflux assay in response to cAMP agonists. Conditions to increase the efficacy of MSC differentiation are currently under investigation. Therefore, we conclude that bone marrow mesenchymal stem cells may have potential applications in treating human airway diseases, such as CF.