The Epstein-Barr Virus Glycoprotein BDLF2 Is Essential for Efficient Viral Spread in Stratified Epithelium.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The ΔBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium. IMPORTANCE The ubiquitous herpesvirus Epstein-Barr virus (EBV) is associated with cancers of B lymphocytes and epithelial cells and is primarily transmitted in saliva. While several models exist for analyzing the life cycle of EBV in B lymphocytes, models of EBV infection in the epithelium have more recently been established. Using an organotypic culture model of epithelium that we previously determined accurately reflects EBV infection in situ, we have ascertained that the loss of the viral envelope protein BDLF2 had little effect on the EBV life cycle in B cells but severely restricted the number of infected cells in organotypic cultures. Loss of BDLF2 has a substantial impact on the size of infected areas, suggesting that BDLF2 plays a specific role in the spread of infection in stratified epithelium.

Histological differentiation of mucus cell subtypes suggests functional compartmentation in the eel esophagus.

We investigated the morphological and histological changes in eel esophagus during the course of freshwater (FW) to seawater (SW) transfer and identified multiple types of mucus cells from tissues that were fixed using Carnoy's solution to retain the mucus structure. The FW esophageal epithelium is stratified and composed of superficial cells, mucus cells, club cells (exocrine cells with a large vacuole), and basal cells. Two types of periodic acid-Schiff (PAS)-positive mucus cells were identified, and they can be further distinguished by the periodic acid-thionin Schiff/KOH/PAS (PAT) method, indicating that C7/9- and C8-sialic acids were produced. Isolectin B4-positive mucus cells were found among the C8-sialic acid-producing cells and located at the tips of the villi at mid-posterior regions of the FW esophagus. The two different muci were immiscible and may form separate layers to protect the tissues from the high osmolality of imbibed SW during early SW acclimation. The densities of club cells and isolectin B4-positive cells decreased after SW acclimation, and cuboidal/columnar epithelial cells subsequently developed for active Na+ and Cl- absorption. Cuboidal/columnar epithelial cells proliferated in scattered array rather than at the bases of the villi, thereby retaining the characteristic of the stratified epithelium. Prominent leukocyte invasion was found at the base of the stratified epithelium at early SW transfer, indicating that the immune system was also activated in response to antigen exposure from imbibed SW. The mucus composition in FW is more complicated than that in SW, fueling further studies for their functions to form unstirred layers as osmoregulatory barriers.

Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells.

BACKGROUND AND AIM: Immune-mediated mucosal inflammation characterized by the release of interleukin (IL)-8 is associated with gastroesophageal reflux disease. ATP released by human esophageal epithelial cells (HEECs) mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including HEECs. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. METHODS: Primary HEECs were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment, and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. RESULTS: Adenosine triphosphate-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of extracellular signal-regulated kinase but not of protein kinase C blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of extracellular signal-regulated kinase, and a P2Y2 receptor antagonist blocked this phosphorylation. CONCLUSIONS: Interleukin-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory gastroesophageal reflux disease.

Differential effect of rebamipide on transmembrane mucin biosynthesis in stratified ocular surface epithelial cells.

Mucins are a group of highly glycosylated glycoproteins responsible for the protection of wet-surfaced epithelia. Recent data indicate that transmembrane mucins differ in their contribution to the protective function of the ocular surface, with MUC16 being the most effective barrier on the apical surface glycocalyx. Here, we investigated the role of the mucoprotective drug rebamipide in the regulation of transmembrane mucin biosynthesis using stratified cultures of human corneal and conjunctival epithelial cells. We find that the addition of rebamipide to corneal, but not conjunctival, epithelial cells increased MUC16 protein biosynthesis. Rebamipide did not affect the levels of MUC1, 4 and 20 compared to control. In these experiments, rebamipide had no effect on the expression levels of Notch intracellular domains, suggesting that the rebamipide-induced increase in MUC16 biosynthesis in differentiated corneal cultures is not regulated by Notch signaling. Overall these findings indicate that rebamipide induces the differential upregulation of MUC16 in stratified cultures of human corneal epithelial cells, which may have implications to the proper restoration of barrier function in ocular surface disease.

The steady state of epidermis: mathematical modeling and numerical simulations.

We consider a model with age and space structure for the epidermis evolution. The model, previously presented and analyzed with respect to the suprabasal epidermis, includes different types of cells (proliferating cells, differentiated cells, corneous cells, and apoptotic cells) moving with the same velocity, under the constraint that the local volume fraction occupied by the cells is constant in space and time. Here, we complete the model proposing a mechanism regulating the cell production in the basal layer and we focus on the stationary case of the problem, i.e. on the case corresponding to the normal status of the skin. A numerical scheme to compute the solution of the model is proposed and its convergence is studied. Simulations are provided for realistic values of the parameters, showing the possibility of reproducing the structure of both "thin" and "thick" epidermis.

Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration.

Expression of the estrogen receptors and steroidogenic enzymes involved in estradiol formation in the monkey vagina.

OBJECTIVE: Estrogens are well recognized to have beneficial effects on vulvovaginal atrophy because of menopause. The distribution of estrogen receptors and enzymes responsible for estradiol (E2) formation within the vagina may provide insight into how dehydroepiandrosterone, a precursor of both estrogens and androgens, improves vulvovaginal atrophy. STUDY DESIGN: The purpose of the study was to determine where the steroidogenic enzymes responsible for E2 formation as well as estrogen receptors are localized in vaginal specimens collected from cynomolgus monkeys (Macaca fascicularis), the closest model to the human. HSD3B1, HSD17B1, HSD17B5, HSD17B12, aromatase (CYP19A1), estrogen receptor (ER)-α, and ER-ß were measured or localized by quantitative real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence. Estrogens were quantified by liquid chromatography/tandem mass spectrometry. RESULTS: All steroidogenic enzymes and estrogen receptors are localized mainly in the superficial layer of the stratified squamous epithelium, blood vessel walls, and muscle fibers of the vagina. Immunolabeling of HSD17B5 and HSD17B12 shows that these enzymes are uniformly distributed from the basal membrane to the superficial keratinized cells, whereas HSD3B1 and aromatase are particularly localized in the outer (external) portion of the epithelial layer. ER-α and ER-ß are also distributed within the vaginal epithelium, with expression especially elevated at the basal membrane level. CONCLUSION: The enzymes responsible for E2 formation as well as ERs are expressed mainly in the superficial layer of the stratified epithelium as well as the muscle layer of the vagina. The present data provide morphologic and biochemical support for the role of local dehydroepiandrosterone transformation into estrogens in regulating epithelial cell maturation, pH, fluid secretion, smooth muscle activity, and blood flow regulation in the primate vagina.

The efficient fabrication of corneal epithelial cell sheets by controlling oxygen concentration.

We have developed a novel method to accelerate the fabrication of epithelial cell sheets by controlling oxygen concentration. Rabbit limbal epithelial cells were proliferated efficiently under hypoxia (2% O2) in comparison to those proliferated under normoxia (20% O2), but were not stratified completely under 2% O2. In contrast, corneal limbal epithelial cells cultured under hypoxia were stratified by re-oxygenation after reaching confluence. Histological and immunofluorescence analyses and colony-forming assays showed that it was possible to fabricate the corneal epithelial cell sheets efficiently by controlling the oxygen concentration. These results indicate that this novel method can be a cost-effective tool for fabricating stratified epithelial cell sheets for corneal regenerative medicine.

Thymidylate synthase (TS) immunostaining in the diagnosis of the myoepithelial cells, basal cells, stratified epithelium cells, and associated tumors.

BACKGROUND: Thymidylate synthase (TS) is an important prognostic biomarker for resistance to 5FU-based adjuvant chemotherapy. Recently, we found that TS was specifically expressed in the nucleus of the myoepithelial cell (MEC), basal cell (BC), transitional epithelial cell (TEC), squamous epithelial cell (SEC), and associated tumor using immunostaining. This prompted us to examine whether TS could be used as a diagnostic biomarker for MEC, BC, TEC, SEC, and associated tumors. METHODS: Formalin-fixed, paraffin-embedded specimens from 186 cases of tumors were immunostaining for expression of TS and p63. The diagnostic capability of TS as a reliable diagnostic marker was evaluated and compared with the expression of p63. RESULTS: TS exhibited a strong specific and stable nuclear immunoreactivity in all specimens including MEC, BC, TEC, and SEC when compared with p63. Notably, a variable degree of TS cytoplasmic positive immunoreactivity was observed in 58.3% of squamous-cell carcinoma (SQCC), 37.5% of basal cell carcinoma (BCC), 44.4% transitional-cell carcinomas (TCC), 41.7% mixed tumor (MT) and 56.5% of adenocarcinoma (ADC) specimens. CONCLUSIONS: In addition to being used as a strong prognostic factor for 5-FU resistance, TS also serves as a promising putative diagnostic marker for identifying MEC, BC, SEC, and TEC from GEC, and for distinguishing SQCC, BCC, TCC, and MT from ADC.

Characterization of the Growth of Chlamydia trachomatis in In Vitro-Generated Stratified Epithelium.

Chlamydia infection targets the mucosal epithelium, where squamous and columnar epithelia can be found. Research on Chlamydia-epithelia interaction has predominantly focused on columnar epithelia, with very little known on how Chlamydia interacts with the squamous epithelium. The stratification and differentiation processes found in the squamous epithelium might influence chlamydial growth and infection dissemination. For this reason, three-dimensional (3D) organotypic stratified squamous epithelial cultures were adapted to mimic the stratified squamous epithelium and chlamydial infection was characterized. Chlamydia trachomatis infection in monolayers and 3D cultures were monitored by immunofluorescence and transmission electron microscopy to evaluate inclusion growth and chlamydial interconversion between elementary and reticulate body. We observed that the stratified epithelium varied in susceptibility to C. trachomatis serovars L2 and D infection. The undifferentiated basal cells were susceptible to infection by both serovars, while the terminally differentiated upper layers were resistant. The differentiating suprabasal cells exhibited different susceptibilities to serovars L2 and D, with the latter unable to establish a successful infection in this layer. Mature elementary body-containing inclusions were much more prevalent in these permissive basal layers, while the uppermost differentiated layers consistently harbored very few reticulate bodies with no elementary bodies, indicative of severely limited bacterial replication and development. For serovar D, the differentiation state of the host cell was a determining factor, as calcium-induced differentiation of cells in a monolayer negatively affected growth of this serovar, in contrast to serovar L2. The apparent completion of the developmental cycle in the basal layers of the 3D cultures correlated with the greater degree of dissemination within and the level of disruption of the stratified epithelium. Our studies indicate that the squamous epithelium is a suboptimal environment for growth, and thus potentially contributing to the protection of the lower genital tract from infection. The relatively more fastidious serovar D exhibited more limited growth than the faster-growing and more invasive L2 strain. However, if given access to the more hospitable basal cell layer, both strains were able to produce mature inclusions, replicate, and complete their developmental cycle.

Generation and Infection of Organotypic Cultures with Epstein-Barr Virus.

While numerous model systems are available to study EBV latency in B cells and have contributed greatly to our understanding of the role of these cells in the viral life cycle, models to study the EBV life cycle in epithelial cells in vitro are lacking. Epithelial cells are poorly infected in vitro, and EBV-infected cell lines have not been successfully obtained from epithelial tumors. Recently, we have demonstrated that organotypic cultures of oral keratinocytes can be used as a model to study EBV infection in the epithelial tissue. These "raft" cultures generate a stratified tissue resembling the epithelium seen in vivo with a proliferating basal layer and differentiating suprabasal layers. Here, we describe generation of EBV-infected raft cultures established from primary oral mucosal epithelial cells, which exhibit high levels of productive replication induced by differentiation, as well as methods to analyze EBV infection.

Serotonin disrupts esophageal mucosal integrity: an investigation using a stratified squamous epithelial model.

BACKGROUND: Serotonin regulates gastrointestinal function, and mast cells are a potential nonneuronal source of serotonin in the esophagus. Tight junction (TJ) proteins in the esophageal epithelium contribute to the barrier function, and the serotonin signaling pathway may contribute to epithelial leakage in gastroesophageal reflux disease. Therefore, the aim of this study was to investigate the role of serotonin on barrier function, TJ proteins, and related signaling pathways. METHODS: Normal primary human esophageal epithelial cells were cultured with use of an air-liquid interface system. Serotonin was added to the basolateral compartment, and transepithelial electrical resistance (TEER) was measured. The expression of TJ proteins and serotonin receptor 7 (5-HT7) was assessed by Western blotting. The involvement of 5-HT7 was assessed with use of an antagonist and an agonist. The underlying cellular signaling pathways were examined with use of specific blockers. RESULTS: Serotonin decreased TEER and reduced the expression of TJ proteins ZO-1, occludin, and claudin 1, but not claudin 4. A 5-HT7 antagonist blocked the serotonin-induced decrease in TEER, and a 5-HT7 agonist decreased TEER. Inhibition of p38 mitogen-activated protein kinase (MAPK) reduced the serotonin-induced decrease in TEER. Inhibition of p38 MAPK blocked the decrease of ZO-1 levels, whereas extracellular-signal-regulated kinase (ERK) inhibition blocked the decrease in occludin levels. Cell signaling pathway inhibitors had no effect on serotonin-induced alterations in claudin 1 and claudin 4 levels. Serotonin induced phosphorylation of p38 MAPK and ERK, and a 5-HT7 antagonist partially blocked serotonin-induced phosphorylation of p38 MAPK but not that of ERK. CONCLUSIONS: Serotonin disrupted esophageal squamous epithelial barrier function by modulating the levels of TJ proteins. Serotonin signaling pathways may mediate the pathogenesis of gastroesophageal reflux disease.

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

Differential expression of ARID3B in normal adult tissue and carcinomas.

ARID3B is a DNA binding protein that is overexpressed in neuroblastoma and ovarian cancer. To understand the extent that ARID3B participates in tumor development, we assessed protein expression of ARID3B in normal adult and malignant tissues. We found that ARID3B is highly expressed in differentiated layers of squamous epithelium. We also examined expression of an alternative splice form of ARID3B and found that it has similar but not identical expression patterns to the full length ARID3B isoform. ARID3B has two closely related paralogues, ARID3A and ARID3C. Each of these 3 family members exhibits different patterns of expression. Of the ARID3 family members, ARID3B is the most widely expressed and is particularly expressed in epithelium. In addition to examining normal tissue, we investigated ARID3B expression in a variety of tumor types. Most notably we found that ARID3B expression is decreased in esophagus and stomach tumors compared to normal corresponding tissues. Our results indicate that the different patterns of ARID3B in normal tissues translate into different roles for ARID3B in carcinomas.

Efficient fabrication of epidermal cell sheets using γ-secretase inhibitor.

BACKGROUND: Epidermal cell sheets have been utilized for regeneration of skin when skin defects occur and prevention of esophageal stricture after endoscopic submucosal dissection. To reduce the cost of cultivation, a novel culture method to shorten a culture process needs to be developed. OBJECTIVES: To shorten a culture process of epidermal cell sheets, we developed a novel culture method to accelerate the fabrication of epidermal cell sheets using γ-secretase inhibitor. METHODS: Normal human epidermal keratinocytes (NHEKs) were cultured using γ-secretase inhibitor, DAPT, during expansion of the cells to confluence and culture without DAPT during stratification. The cell growth, quantitative gene expression of stem/progenitor or differentiation markers, and protein expression of these markers were analyzed to verify the effectiveness of the novel method. RESULTS: The proliferation of NHEKs on cell-culture inserts was promoted using DAPT. However, NHEKs were not stratified completely in the presence of DAPT. In contrast, NHEKs cultured using DAPT were stratified and differentiated by eliminating the inhibitor after the cells reached confluence. Real-time PCR analyses showed that the gene expressions of putative epithelial stem/progenitor cell markers and epidermis differentiation markers in the cell sheets fabricated using this novel method were significantly higher than those in the cell sheets fabricated without DAPT. Histological and immunofluorescence analyses revealed that it was possible to fabricate well-differentiated epidermal cell sheets efficiently by the novel culture method. The culture period was shortened to 67% of the time required for the control group. In feeder-free conditions, stratified epidermal cell sheets were also fabricated using DAPT. CONCLUSIONS: The novel culture method using γ-secretase inhibitor, DAPT, was found to be effective for fabricating epidermal cell sheets.

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.