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1.  Protein-Coated Nanoparticles Are Internalized by the Epithelial Cells of the Female Reproductive Tract and Induce Systemic and Mucosal Immune Responses 
PLoS ONE  2014;9(12):e114601.
The female reproductive tract (FRT) includes the oviducts (fallopian tubes), uterus, cervix and vagina. A layer of columnar epithelium separates the endocervix and uterus from the outside environment, while the vagina is lined with stratified squamous epithelium. The mucosa of the FRT is exposed to antigens originating from microflora, and occasionally from infectious microorganisms. Whether epithelial cells (ECs) of the FRT take up (sample) the lumen antigens is not known. To address this question, we examined the uptake of 20–40 nm nanoparticles (NPs) applied vaginally to mice which were not treated with hormones, epithelial disruptors, or adjuvants. We found that 20 and 40 nm NPs are quickly internalized by ECs of the upper FRT and within one hour could be observed in the lymphatic ducts that drain the FRT, as well as in the ileac lymph nodes (ILNs) and the mesenteric lymph nodes (MLNs). Chicken ovalbumin (Ova) conjugated to 20 nm NPs (NP-Ova) when administered vaginally reaches the internal milieu in an immunologically relevant form; thus vaginal immunization of mice with NP-Ova induces systemic IgG to Ova antigen. Most importantly, vaginal immunization primes the intestinal mucosa for secretion of sIgA. Sub-cutaneous (s.c) boosting immunization with Ova in complete Freund's adjuvant (CFA) further elevates the systemic (IgG1 and IgG2c) as well as mucosal (IgG1 and sIgA) antibody titers. These findings suggest that the modes of antigen uptake at mucosal surfaces and pathways of antigen transport are more complex than previously appreciated.
doi:10.1371/journal.pone.0114601
PMCID: PMC4260873  PMID: 25490456
2.  Progressive Proximal-to-Distal Reduction in Expression of the Tight Junction Complex in Colonic Epithelium of Virally-Suppressed HIV+ Individuals 
PLoS Pathogens  2014;10(6):e1004198.
Effective antiretroviral therapy (ART) dramatically reduces AIDS-related complications, yet the life expectancy of long-term ART-treated HIV-infected patients remains shortened compared to that of uninfected controls, due to increased risk of non-AIDS related morbidities. Many propose that these complications result from translocated microbial products from the gut that stimulate systemic inflammation – a consequence of increased intestinal paracellular permeability that persists in this population. Concurrent intestinal immunodeficiency and structural barrier deterioration are postulated to drive microbial translocation, and direct evidence of intestinal epithelial breakdown has been reported in untreated pathogenic SIV infection of rhesus macaques. To assess and characterize the extent of epithelial cell damage in virally-suppressed HIV-infected patients, we analyzed intestinal biopsy tissues for changes in the epithelium at the cellular and molecular level. The intestinal epithelium in the HIV gut is grossly intact, exhibiting no decreases in the relative abundance and packing of intestinal epithelial cells. We found no evidence for structural and subcellular localization changes in intestinal epithelial tight junctions (TJ), but observed significant decreases in the colonic, but not terminal ileal, transcript levels of TJ components in the HIV+ cohort. This result is confirmed by a reduction in TJ proteins in the descending colon of HIV+ patients. In the HIV+ cohort, colonic TJ transcript levels progressively decreased along the proximal-to-distal axis. In contrast, expression levels of the same TJ transcripts stayed unchanged, or progressively increased, from the proximal-to-distal gut in the healthy controls. Non-TJ intestinal epithelial cell-specific mRNAs reveal differing patterns of HIV-associated transcriptional alteration, arguing for an overall change in intestinal epithelial transcriptional regulation in the HIV colon. These findings suggest that persistent intestinal epithelial dysregulation involving a reduction in TJ expression is a mechanism driving increases in colonic permeability and microbial translocation in the ART-treated HIV-infected patient, and a possible immunopathogenic factor for non-AIDS related complications.
Author Summary
While antiretroviral therapy for HIV-infected patients is remarkably effective in suppressing viral replication and preventing progression to AIDS, treated patients still have a shorter life expectancy due to increased risks for non-AIDS associated morbidities. Recent data showed that these complications are associated with chronic systemic inflammation, and it is hypothesized that bacterial products breaching the intestinal barrier may cause the inflammation. It is known that HIV induces persistent intestinal mucosal immunodeficiency, but evidence for structural damage to the intestinal epithelium is lacking in the antiretroviral-treated patient population. Here, we characterized the intestinal epithelial damage that leads to increased intestinal permeability in this population. We found that while the colonic epithelial layer is intact microscopically, intercellular tight junctions (TJ) are down-regulated at the transcriptional and translational levels. We observed further that TJ transcripts progressively decrease along the proximal-to-distal HIV gut. Concurrent alterations in the levels of non-TJ epithelial transcripts suggest that epithelial cells in the HIV gut are transcriptionally dysregulated. Our data provide evidence that TJ disruption is a novel mechanism for increasing colonic permeability in the antiretroviral-treated HIV patient, which may then result in systemic inflammation and associated complications.
doi:10.1371/journal.ppat.1004198
PMCID: PMC4072797  PMID: 24968145
3.  HIV gp120 Binds to Mannose Receptor on Vaginal Epithelial Cells and Induces Production of Matrix Metalloproteinases 
PLoS ONE  2011;6(11):e28014.
Background
During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR) as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs) downstream of HIV gp120 binding to hMR.
Principal Findings
Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line) and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody.
Conclusion
hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the vaginal epithelium.
doi:10.1371/journal.pone.0028014
PMCID: PMC3222676  PMID: 22132194
4.  The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium 
The Journal of Cell Biology  1990;111(3):1255-1263.
The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.
PMCID: PMC2116287  PMID: 2202736
5.  Listeria monocytogenes Invades the Epithelial Junctions at Sites of Cell Extrusion 
PLoS Pathogens  2006;2(1):e3.
Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A and the host protein E-cadherin, located below the epithelial tight junctions at the lateral cell-to-cell contacts. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes breaches the tight junctions to gain access to this basolateral receptor protein. We determined that L. monocytogenes does not actively disrupt the tight junctions, but finds E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are expelled and detached from the epithelium by extrusion. The surrounding cells reorganize to form a multicellular junction that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at multicellular junctions during and after cell extrusion, and that L. monocytogenes takes advantage of junctional remodeling to adhere to and subsequently invade the epithelium. In intact epithelial monolayers, an anti-E-cadherin antibody specifically decorates multicellular junctions and blocks L. monocytogenes adhesion. Furthermore, an L. monocytogenes mutant in the Internalin A gene is completely deficient in attachment to the epithelial apical surface and is unable to invade. We hypothesized that L. monocytogenes utilizes analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the junctions at the cell extrusion zone of villus tips are the specific target for L. monocytogenes adhesion and invasion. Thus, L. monocytogenes exploits the dynamic nature of epithelial renewal and junctional remodeling to breach the intestinal barrier.
Synopsis
Studies in microbial pathogenesis are just beginning to address how epithelial cell polarity and host tissue architecture influence host-pathogen interactions. It has long been known that Listeria monocytogenes invasion of epithelial cells requires binding of host cell E-cadherin, a cell-to-cell junction protein. However, an ongoing question has been how L. monocytogenes reaches this receptor, since E-cadherin is located in the basolateral membrane of enterocytes and is inaccessible to bacteria in the intestinal lumen. By examining polarized epithelial monolayers in tissue culture and rabbit intestine in vivo, the authors investigate how and where L. monocytogenes breaches the tight junctions to interact with E-cadherin and invade. They show that L. monocytogenes takes advantage of a temporal window of junctional reorganization that exposes E-cadherin at sites where senescent cells are extruded and removed from the epithelium. In the small intestine, cell extrusion is confined to the tips of the intestinal villi, and the authors show that junctions at the villus tips are the sites of L. monocytogenes invasion in vivo. If basolateral proteins are exposed at cell extrusion sites, do other microbes that utilize basolateral cellular receptors also find them?
doi:10.1371/journal.ppat.0020003
PMCID: PMC1354196  PMID: 16446782
6.  Commensal Bacteria Modulate Innate Immune Responses of Vaginal Epithelial Cell Multilayer Cultures 
PLoS ONE  2012;7(3):e32728.
The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives.
doi:10.1371/journal.pone.0032728
PMCID: PMC3296736  PMID: 22412914
7.  Human beta-defensin-1: an antimicrobial peptide of urogenital tissues. 
Journal of Clinical Investigation  1998;101(8):1633-1642.
Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.
PMCID: PMC508744  PMID: 9541493
8.  Early HIV-1 Target Cells in Human Vaginal and Ectocervical Mucosa 
After translocation through the pleuristratified epithelium of the lower female genital tract, HIV-1 encounters potential target mononuclear cells in the lamina propria of the vagina and ectocervix. Here we show that each major type of genital mononuclear cell, including dendritic cells (DCs), macrophages and lymphocytes, are susceptible to HIV-1 in vitro. Among suspensions of vaginal and ectocervical mononuclear cells, DCs were the first cells to take up virus, containing GFP-tagged virions as early as 15 minutes after exposure. At 2 hr after exposure, DCs still contained the largest proportion of infected cells compared to lamina propria macrophages and lymphocytes from the same mucosal compartment. By 4 days, however, lymphocytes from both vaginal and ectocervical mucosa supported the highest level of HIV-1 replication. Genital macrophages from the same mucosal tissues also were permissive to HIV-1, in sharp contrast to intestinal macrophages, which we previously have shown do not support HIV-1 replication. Thus, among human vaginal and ectocervical mononuclear target cells, DCs are the first to take up HIV-1 and T cells support the most robust viral replication. Further characterization of the parameters of HIV-1 infection in genital mononuclear cells will enlarge our understanding of HIV-1 infection in the female genital tract.
doi:10.1111/j.1600-0897.2010.00939.x
PMCID: PMC3077123  PMID: 21118402
Dendritic cells; entry; genital; lymphocytes; macrophages; replication
9.  Expression of JAM-A in the Human Corneal Endothelium and Retinal Pigment Epithelium: Localization and Evidence for Role in Barrier Function 
Purpose
Junctional adhesion molecules (JAMs) are a family of adhesion proteins found in intercellular junctions. Evidence suggests that JAM-A is important for the regulation of tight junction assembly and epithelial barrier function. The authors recently reported that JAM-A is expressed in rabbit corneal endothelium and that antibody to JAM-A produces corneal swelling. In the present study, they investigate JAM-A expression in the human corneal endothelium and retinal pigment epithelium (RPE) and examine the effect of a function-blocking antibody to JAM-A on the permeability of cultured RPE cell monolayers.
Methods
Expression of JAM-A in human corneal endothelium, human RPE tissue, and cultured ARPE-19 monolayers was assessed by immunofluorescence confocal microscopy. Localization of JAM-A was compared with the tight junction-associated protein zonula occludens-1 (ZO-1). To investigate JAM-A function in ARPE-19 cells, ARPE-19 monolayers were subjected to a calcium switch protocol to disrupt cell junctions and treated with a function-blocking antibody to JAM-A or an isotype-matched control. Dextran flux assays were performed to assess the effect of JAM-A antibody on ARPE-19 monolayer permeability.
Results
Expression of JAM-A was observed in human corneal endothelium, and its distribution correlated with the tight junction-associated protein ZO-1. In addition, expression of JAM-A was observed in human RPE and in intercellular junctions of ARPE-19 monolayers. The localization pattern of JAM-A in the RPE and ARPE-19 monolayers was similar to that of ZO-1. ARPE-19 monolayers treated with antibody to JAM-A demonstrated a 33% increase in permeability to 10,000 MWt dextran compared with monolayers treated with control antibody.
Conclusions
Results of this study provide new information about JAM-A expression in tight junctions of the human corneal endothelium and human RPE. The observation that antibodies to JAM-A increase ARPE-19 monolayer permeability is consistent with previous findings of JAM-A function in epithelial tight junctions and suggests JAM-A may have a role in the regulation of RPE barrier function.
doi:10.1167/iovs.06-1536
PMCID: PMC2074894  PMID: 17724169
10.  The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium 
The Journal of Cell Biology  1989;109(6):2817-2832.
Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.
PMCID: PMC2115929  PMID: 2592406
11.  The Neonatal Fc Receptor (FcRn) Enhances Human Immunodeficiency Virus Type 1 (HIV-1) Transcytosis across Epithelial Cells 
PLoS Pathogens  2013;9(11):e1003776.
The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure.
Author Summary
HIV-1 causes a sexually transmitted disease. However, the mechanisms employed by the virus to cross genital tract tissue and establish infection are uncertain. Since cervicovaginal fluid is acidic and HIV-1 in cervicovaginal fluid is likely coated with antibodies, we explored the effect of low pH and HIV-1-specific antibodies on transcytosis, the movement of HIV-1 across tight-junctioned epithelial cells. We found that the combination of HIV-1-specific antibodies and low pH enhanced transcytosis as much as 20-fold. Virus that underwent transcytosis under these conditions was infectious, and infectivity was highly influenced by whether or not the antibody neutralized the virus. We observed enhanced transcytosis using antibody from cervicovaginal and seminal fluids and using transmitted/founder strains of HIV-1. We also found that the enhanced transcytosis was due to the Fc neonatal receptor (FcRn), which binds immune complexes at acidic pH and releases them at neutral pH. Finally, staining of human tissue revealed abundant FcRn expression on columnar epithelial cells of penile urethra and endocervix. Our findings reveal a novel mechanism wherein HIV-1 may facilitate its own transmission by usurping the antibody response directed against itself. These results have important implications for HIV vaccine development and for understanding the earliest events in HIV transmission.
doi:10.1371/journal.ppat.1003776
PMCID: PMC3836734  PMID: 24278022
12.  The claudin family of proteins in human malignancy: a clinical perspective 
Tight junctions, or zonula occludens, are the most apical component of the junctional complex and provide one form of cell–cell adhesion in epithelial and endothelial cells. Nearly 90% of malignant tumors are derived from the epithelium. Loss of cell–cell adhesion is one of the steps in the progression of cancer to metastasis. At least three main tight junction family proteins have been discovered: occludin, claudin, and junctional adhesion molecule (JAM). Claudins are the most important structural and functional components of tight junction integral membrane proteins, with at least 24 members in mammals. They are crucial for the paracellular flux of ions and small molecules. Overexpression or downregulation of claudins is frequently observed in epithelial-derived cancers. However, molecular mechanisms by which claudins affect tumorigenesis remain largely unknown. As the pivotal proteins in epithelial cells, altered expression and distribution of different claudins have been reported in a wide variety of human malignancies, including pancreatic, colonic, lung, ovarian, thyroid, prostate, esophageal, and breast cancers. In this review, we will give the readers an overall picture of the changes in claudin expression observed in various cancers and their mechanisms of regulation. Downregulation of claudins contributes to epithelial transformation by increasing the paracellular permeability of nutrients and growth factors to cancerous cells. In the cases of upregulation of claudin expression, the barrier function of the cancerous epithelia changes, as they often display a disorganized arrangement of tight junction strands with increased permeability to paracellular markers. Finally, we will summarize the literature suggesting that claudins may become useful biomarkers for cancer detection and diagnosis as well as possible therapeutic targets for cancer treatment.
doi:10.2147/CMAR.S38294
PMCID: PMC3825674  PMID: 24232410
tight junctions; claudins; human cancers
13.  Characterization of low molecular weight antimicrobial peptide from human female reproductive tract 
Background & objectives:
The mechanisms that protect female upper genital tract from ascending infection by microbes present in vagina are only partially understood. It is expected that epithelial cells in mucosal surfaces and their secretions directly interfere with microbial colonization and invasion. This study was aimed to demonstrate the expression of 2 kDa antimicrobial peptide which was identified and purified from female genital tract tissues using chromatographic techniques.
Methods:
Low molecular weight proteins were isolated from human female reproductive tract tissues obtained from premenopausal women. Antimicrobial activity of these LMW proteins was assessed against different reproductive tract pathogens viz., Neisseria gonorrhoeae, Group B streptococcus, Gardnerella vaginalis, Escherechia coli and Candida albicans. The expression of these peptides were also documented in reproductive tract tissues with the help of hyperimmune sera raised against the rabbits. The purified peptide was characterized by N-terminal sequencing.
Results:
Immunohistochemical and immunofluorescence studies demonstrated that 2 kDa peptide was expressed in the stratified squamous epithelial cells of the ectocervix while it was absent in columnar epithelial cells of upper genital tract. Upregulation of the expression of this peptide was observed in patients of chronic non-specific cervicitis and acute on chronic cervicitis. This purified antimicrobial peptide also showed broad spectrum antimicrobial activity against different reproductive tract pathogens.
Interpretation & conclusions:
Considering the emerging bacterial resistance against conventional antibiotics, isolation and understanding of the expression of antimicrobial peptides from female reproductive tissue extracts may provide some leads towards the development of strategies for the treatment of reproductive tract infections.
doi:10.4103/0971-5916.90996
PMCID: PMC3249967  PMID: 22199108
Antimicrobials; antimicrobial peptides; female reproductive tract; innate immunity; sexually transmitted infection
14.  Inflammation and Disruption of the Mucosal Architecture in Claudin-7-Deficient Mice 
Gastroenterology  2011;142(2):305-315.
Background & Aims
Integrity of the intestinal epithelium is required for nutrition absorption and defense against pathogens. Claudins are cell adhesion molecules that localize at tight junctions (TJs); many are expressed in the intestinal tract, but little is known about their functions. Claudin-7 is unique in that it has a stronger basolateral membrane distribution than other claudins, which localize primarily to apical TJs in the intestinal epithelium. We investigated the basolateral functions of claudin-7 and assessed the effects of disruption of Cldn7 in intestines of mice.
Methods
We generated Cldn7−/− mice and examined their intestines by histology, molecular and cellular biology, and biochemistry approaches. We carried out gene silencing experiments in epithelial cell lines using small interfering (si)RNAs.
Results
The Cldn7−/− mice had severe intestinal defects that included mucosal ulcerations, epithelial cell sloughing, and inflammation. Intestines of Cldn7−/− mice produced significantly higher levels of cytokines, the NF-κB p65 subunit, and COX-2; they also upregulated expression of matrix metalloproteinases (MMPs)-3 and -7. siRNA in epithelial cell lines demonstrated that the increased expression of MMP-3 resulted directly from claudin-7 depletion, whereas that of MMP-7 resulted from inflammation. Electron microscopy analysis showed that intestines of Cldn7−/− mice had intercellular gaps below TJs and cell-matrix loosening. Deletion of Cldn7 reduced expression and altered localization of the integrin α2 subunit; disrupted formation of complexes of claudin-7, integrin α2, and claudin-1 that normally form in epithelial basolateral compartments of intestines.
Conclusion
In mice, claudin-7 has non-TJ functions, including maintenance of epithelial cell–matrix interactions and intestinal homeostasis.
doi:10.1053/j.gastro.2011.10.025
PMCID: PMC3267838  PMID: 22044670
Mucosal integrity; epithelial barrier; mouse model; permeability; IBD
15.  House Dust Mite Der p 1 Effects on Sinonasal Epithelial Tight Junctions 
Background
Epithelial permeability is highly dependent upon the integrity of tight junctions, cell-cell adhesion complexes located at the apical aspect of the lateral membrane of polarized epithelial cells. We hypothesize that sinonasal epithelial exposure to Der p 1 house dust mite antigen decreases expression of tight junction proteins (TJPs), representing a potential mechanism for increased permeability and presentation of antigens across the sinonasal epithelial layer.
Methods
Confluent cultured primary human sinonasal epithelial cells were exposed to recombinant Der p 1 antigen versus control, and transepithelial resistance measurements were performed over 24 hours. Antibody staining for a panel of tight junction proteins was examined with immunofluorescence/confocal microscopy and Western blotting. Tissue for these experiments was obtained from 4 patients total.
Results
Der p 1 exposed sinonasal cells showed a marked decrease in transepithelial resistance when compared to control cells. In addition, results of Western immunoblot and immunofluorescent labeling demonstrated decreased expression of TJPs claudin-1 and junction adhesion molecule-A (JAM-A) in Der p 1 exposed cultured sinonasal cells versus controls.
Conclusion
Der p 1 antigen exposure decreases sinonasal epithelium TJP expression, most notably seen in JAM-A and claudin-1 in these preliminary experiments. This decreased TJP expression likely contributes to increased epithelial permeability and represents a potential mechanism for transepithelial antigen exposure in allergic rhinitis.
doi:10.1002/alr.21168
PMCID: PMC3782710  PMID: 23592402
House dust mite; rhinitis; tight junction; allergic rhinitis; epithelium; sinonasal; junction adhesion molecule; claudin-1
16.  Epithelial Permeability Alterations in an In Vitro Air-Liquid Interface Model of Allergic Fungal Rhinosinusitis 
Background
Chronic rhinosinusitis (CRS) is an inflammatory upper-airway disease with numerous etiologies. Patients with a characteristic subtype of CRS, allergic fungal rhinosinusitis (AFRS), display increased expression of Th2 cytokines and antigen-specific IgE. Various sinonasal inflammatory conditions are associated with alterations in epithelial barrier function. The aim of this study was to compare epithelial permeability and intercellular junctional protein expression amongst cultured primary sinonasal cells from AFRS patients versus non-inflammatory controls.
Methods
Epithelial cells isolated from paranasal sinus mucosa of AFRS and non-inflammatory control patients were grown to confluence on permeable supports and transitioned to air-liquid interface (ALI). Trans-epithelial resistance (TER) was measured with a horizontal Ussing chamber to characterize the functional permeability of each cell type. After TER recordings were complete, a panel of intercellular junctional proteins was assessed by Western blot and immunofluorescence labeling followed by confocal microscopy.
Results
After 12 samples were measured from each group, we observed a 41% mean decrease in TER in AFRS cells (296±89 ohms × cm2) compared to control (503±134 ohms × cm2, P=0.006). TER deficits observed in AFRS were associated with decreased expression of the tight junction proteins occludin and Junctional Adhesion Molecule-A (JAM-A), and increased expression of a leaky tight junction protein claudin-2.
Conclusions
Cultured sinonasal epithelium from AFRS patients displayed increased epithelial permeability and altered expression of intercellular junctional proteins. Given that these cells were not incubated with inflammatory cytokines in vitro, the cultured AFRS epithelial alterations may represent a retained modification in protein expression from the in vivo phenotype.
doi:10.1002/alr.21077
PMCID: PMC3511593  PMID: 22927233
Adherens junction; allergic fungal rhinosinusitis; allergic rhinitis; claudin-2; E-cadherin; epithelial permeability; junction adhesion molecule-A; occludin; sinonasal epithelium; tight junction
17.  The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex 
The Journal of Cell Biology  1988;107(4):1575-1587.
The role of the epithelial adhesion molecule uvomorulin in the formation of the epithelial junctional complex in the Madin-Darby canine kidney (MDCK) cell line was investigated. Experiments were carried out to determine whether specific inhibition of uvomorulin function would interfere selectively with the formation, stability, or function of the apical zonula adherens (ZA) and zonula occludens (ZO), or whether it would interfere with all forms of intercellular contact including the desmosomes. The effects of blocking antibodies and Fab fragments to uvomorulin on the formation of the junctional complex was examined with a Ca2+ switch assay for de novo junction assembly. The formation of the ZO, the ZA, and the desmosomes was assayed by fluorescence staining with an antibody to the tight junction-specific protein ZO-1, with rhodamine-phalloidin for ZA-associated actin filaments, and with an anti-desmoplakin antibody, respectively. Under different conditions and times of antibody treatment the extent of inhibition of the formation of each of the junctional elements was very similar. The ability of the cells to eventually overcome the inhibitory effect of the antibodies and form junctions correlated with the reappearance of uvomorulin at the regions of cell-cell contact. Therefore uvomorulin seems to mediate an early adhesion event between epithelial cells that is a prerequisite for the assembly of all elements of the junctional complex. In contrast, the transepithelial electrical resistance of confluent, well-established monolayers of MDCK cells grown on filters was not greatly affected by treatment with the various antibodies or Fab fragments. A small transient decrease in resistance observed with the polyclonal alpha-uvomorulin IgG may be due to a more subtle modulation of the junctional complex.
PMCID: PMC2115263  PMID: 3049625
18.  The suprabasal layer of corneal epithelial cells represents the major barrier site to the passive movement of small molecules and trafficking leukocytes 
Aims
To investigate the site of barrier function to the passive diffusion of a small molecule (phalloidin) in the corneal epithelium in the mouse.
Methods
Penetration of phalloidin (molecular weight 1115 daltons) into the cornea was evaluated by studying fluorescent binding of phalloidin to actin in tissue sections, in whole mount preparations, and in the fixed intact globe by confocal microscopy. In addition, the location of tight junction proteins in the individual layers of the corneal epithelium was determined by immunohistochemistry.
Results
Phalloidin staining of corneal sections was positive in all corneal layers in tissue sections and in all layers of the corneal epithelium except the suprabasal layer in excised fixed whole mounts of the cornea. However, when phalloidin staining was attempted in intact fixed globes, before excision of the cornea for whole mount preparation, only the most superficial layer of cells was stained indicating that phalloidin could not penetrate the tissue beyond the suprabasal epithelial layer. Detergent (Triton X‐100) treatment of the excised cornea and the intact fixed globe, allowed penetration of phalloidin into the suprabasal epithelial layer. Tight junction proteins occludin, ZO‐1 and claudin were present in most layers of the cornea but while ZO‐1 and occludin were distributed in a typical pericellular pattern, claudin seemed to be particularly prominent in the suprabasal layer and appeared only as a discontinuous punctate pericellular pattern in the superficial layer. Intraepithelial leukocytes were detected in the superficial epithelium and the basal epithelium but not in the suprabasal epithelium.
Conclusion
The suprabasal epithelium cell layer appears to represent the main barrier site to the passage of small molecules and cells in the mouse cornea and this property may be attribuatable to prominent claudin expression in this layer.
doi:10.1136/bjo.2006.097188
PMCID: PMC1857692  PMID: 17020902
19.  JUNCTIONAL COMPLEXES IN VARIOUS EPITHELIA 
The Journal of Cell Biology  1963;17(2):375-412.
The epithelia of a number of glands and cavitary organs of the rat and guinea pig have been surveyed, and in all cases investigated, a characteristic tripartite junctional complex has been found between adjacent cells. Although the complex differs in precise arrangement from one organ to another, it has been regularly encountered in the mucosal epithelia of the stomach, intestine, gall bladder, uterus, and oviduct; in the glandular epithelia of the liver, pancreas, parotid, stomach, and thyroid; in the epithelia of pancreatic, hepatic, and salivary ducts; and finally, between the epithelial cells of the nephron (proximal and distal convolution, collecting ducts). The elements of the complex, identified as zonula occludens (tight junction), zonula adhaerens (intermediary junction), and macula adhaerens (desmosome), occupy a juxtaluminal position and succeed each other in the order given in an apical-basal direction. The zonula occludens (tight junction) is characterized by fusion of the adjacent cell membranes resulting in obliteration of the intercellular space over variable distances. Within the obliterated zone, the dense outer leaflets of the adjoining cell membranes converge to form a single intermediate line. A diffuse band of dense cytoplasmic material is often associated with this junction, but its development varies from one epithelium to another. The zonula adhaerens (intermediate junction) is characterized by the presence of an intercellular space (∼200 A) occupied by homogeneous, apparently amorphous material of low density; by strict parallelism of the adjoining cell membranes over distances of 0.2 to 0.5 µ; and by conspicuous bands of dense material located in the subjacent cytoplasmic matrix. The desmosome or macula adhaerens is also characterized by the presence of an intercellular space (∼240 A) which, in this case, contains a central disc of dense material; by discrete cytoplasmic plaques disposed parallel to the inner leaflet of each cell membrane; and by the presence of bundles of cytoplasmic fibrils converging on the plaques. The zonula occludens appears to form a continuous belt-like attachment, whereas the desmosome is a discontinuous, button-like structure. The zomula adhaerens is continuous in most epithelia but discontinuous in some. Observations made during experimental hemoglobinuria in rats showed that the hemoglobin, which undergoes enough concentration in the nephron lumina to act as an electron-opaque mass tracer, does not penetrate the intercellular spaces beyond the zonula occludens. Similar observations were made in pancreatic acini and ducts where discharged zymogen served as a mass tracer. Hence the tight junction is impervious to concentrated protein solutions and appears to function as a diffusion barrier or "seal." The desmosome and probably also the zonula adhaerens may represent intercellular attachment devices.
PMCID: PMC2106201  PMID: 13944428
20.  Distinct Proinflammatory Host Responses to Neisseria gonorrhoeae Infection in Immortalized Human Cervical and Vaginal Epithelial Cells 
Infection and Immunity  2001;69(9):5840-5848.
In this study we utilized immortalized morphologically and functionally distinct epithelial cell lines from normal human endocervix, ectocervix, and vagina to characterize gonococcal epithelial interactions pertinent to the lower female genital tract. Piliated, but not nonpiliated, N. gonorrhoeae strain F62 variants actively invaded these epithelial cell lines, as demonstrated by an antibiotic protection assay and confocal microscopy. Invasion of these cells by green fluorescent protein-expressing gonococci was characterized by colocalization of gonococci with F actin, which were initially detected 30 min postinfection. In all three cell lines, upregulation of interleukin 8 (IL-8) and IL-6, intercellular adhesion molecule 1 (CD54), and the nonspecific cross-reacting antigen (CD66c) were detected 4 h after infection with piliated and nonpiliated gonococci. Furthermore, stimulation of all three cell lines with gonococcal whole-cell lysates resulted in a similar upregulation of IL-6 and IL-8, confirming that bacterial uptake is not essential for this response. Increased levels of IL-1 were first detected 8 h after infection with gonococci, suggesting that the earlier IL-8 and IL-6 responses were not mediated through the IL-1 signaling pathway. The IL-1 response was limited to cultures infected with piliated gonococci and was more vigorous in the endocervical epithelial cells. The ability of gonococci to stimulate distinct proinflammatory host responses in these morphologically and functionally different compartments of the lower female genital tract may contribute directly to the inflammatory signs and symptoms characteristic of disease caused by N. gonorrhoeae.
doi:10.1128/IAI.69.9.5840-5848.2001
PMCID: PMC98702  PMID: 11500462
21.  Intestinal Barrier Function: Molecular Regulation and Disease Pathogenesis 
The intestinal epithelium is a single-cell layer that constitutes the largest and most important barrier against the external environment. It acts as a selectively permeable barrier permitting the absorption of nutrients, electrolytes and water, while maintaining an effective defense against intraluminal toxins, antigens and enteric flora. The epithelium maintains its selective barrier function through the formation of complex protein-protein networks that mechanically link adjacent cells and seal the intercellular space. The protein networks connecting epithelial cells form three adhesive complexes: desmosomes, adherens junctions and tight junctions. These complexes consist of transmembrane proteins that interact extracellularly with adjacent cells and intracellularly with adaptor proteins that link to the cytoskeleton. Over the past decade, there has been increasing recognition of an association between disrupted intestinal barrier function and the development of autoimmune and inflammatory diseases. In this review, we summarize the evolving understanding of the molecular composition and regulation of intestinal barrier function. We discuss the interactions between innate and adaptive immunity and intestinal epithelial barrier function, as well as the impact of exogenous factors on intestinal barrier function. Finally, we summarize clinical and experimental evidence demonstrating intestinal epithelial barrier dysfunction as a major factor contributing to the predisposition to inflammatory diseases including food allergy, inflammatory bowel diseases and celiac disease.
doi:10.1016/j.jaci.2009.05.038
PMCID: PMC4266989  PMID: 19560575
22.  Association of mesenchymal cells and immunoglobulins with differentiating epithelial cells 
Background
Mesenchymal-epithelial interactions play an important role in the physiology and pathology of epithelial tissues. Mesenchymal cells either associate with epithelium basement membrane [pericytes and perivascular monocyte-derived cells (MDC)] or reside within epithelium (MDC and T cells). Although intraepithelial mesenchymal cells were suggested to contribute to the epithelium physiology, their association with particular steps in differentiation of epithelial cells, interactions among themselves, and their fate remain unclear. We studied epitopes of mesenchymal cells and their products (immunoglobulins) in stratified epithelium of uterine ectocervix, which is one of the prototypes of complete cellular differentiation from stem into the aged cells.
Results
Perivascular CD14 primitive MDC associated with basal (stem) epithelial cells. Thy-1 pericytes of microvasculature secreted intercellular vesicles, which associated with Ki67 postmitotic epithelial cells expressing MHC class I. Intraepithelial T cells showed an association with veiled type MDC [dendritic cell (DC) precursors] among parabasal cells, and exhibited fragmentation after entering intermediate (mature) epithelial layers. Mature DC secreted CD68 and exhibited fragmentation after reaching mid intermediate layers. Binding of IgM was detected at the top of each layer: in the upper parabasal, upper intermediate, and most surface epithelial cells. IgG was confined to the entire superficial layer.
Conclusions
These data suggest that the phylogenetically and ontogenetically developed hierarchy of mesenchymal cells (MDC, pericytes, T cells) and immunoglobulins (IgM, IgG) accompanies differentiation of epithelial cells from immature into the mature and aged phenotype. Further studies of an involvement of mesenchymal cells in the regulation of tissue homeostasis may bring novel approaches to the prevention and therapy of tissue dysfunctions characterized by permanent tissue immaturity (muscular dystrophy) or accelerated aging (degenerative diseases).
doi:10.1186/1471-213X-1-11
PMCID: PMC34117  PMID: 11439174
23.  CELL JUNCTIONS IN AMPHIBIAN SKIN 
The Journal of Cell Biology  1965;26(1):263-291.
Cell junctions have been investigated in the amphibian epidermis, a stratified squamous epithelium, and compared to those described previously in simple columnar epithelia of mammalian cavitary organs. In adult frogs and toads, and in larvae approaching metamorphosis, belts of membrane fusion or zonulae occludentes of considerable depth are regularly found between adjoining cells of the outermost layer of the stratum corneum, binding the cells together into a continuous, uninterrupted sheet. Another set of occluding zonules appears in the second cornified layer (when such a layer is present), and a third set usually occurs in the outermost layer of the stratum granulosum. Specialized elements described as "modified" and "composite" desmosomes are encountered along the lateral and basal aspects, respectively, of the cornified cells; ordinary desmosomes and maculae occludentes (i.e., spots of membrane fusion) are found in all other strata. The usual 200 A intercellular gap is generally maintained between the cells of the stratum germinativum at the basal ends of the intercellular spaces. Hence, the intercellular spaces of the epidermis form a largely continuous network, closed to the external medium and open to the dermal interstitia. The situation is comparable to that found in columnar epithelia, except that the intercellular spaces are much more extensive, and an extracellular subcompartment (or two) apparently exists in the stratum corneum and between the latter and the stratum granulosum. The last subcompartment is usually filled with a dense substance, probably derived from discharged secretory granules. The tripartite junctional complex characteristic of lumen-lining epithelia (i.e., a zonula occludens followed by a zonula adhaerens, and desmosome) is seen only in early larvae; in adults and in larvae approaching metamorphosis, the occluding zonule is followed directly by a series of modified desmosomes. Interpreted in the light of current physiological data, these findings suggest that the diffusion of water, ions, and small, water-soluble molecules is impeded along the intercellular spaces of the epidermis by zonulae occludentes while it is facilitated from cell to cell within the epidermis by zonulae and maculae occludentes.
PMCID: PMC2106698  PMID: 5859021
24.  Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells 
Cell and Tissue Research  2013;353(1):99-115.
Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions (“tessellate junctions”), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker.
doi:10.1007/s00441-013-1645-3
PMCID: PMC3691483  PMID: 23689684
Adhering junctions; Desmosomes; Tessellate junctions; PERP; Tetraspanins; Immunocytochemistry
25.  Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical- basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein 
The Journal of Cell Biology  1996;134(4):1031-1049.
Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.
PMCID: PMC2120963  PMID: 8769425

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