Showing papers on "Epithelium published in 2023"

Notch1 mutations drive clonal expansion in normal esophageal epithelium but impair tumor growth

Abstract: NOTCH1 mutant clones occupy the majority of normal human esophagus by middle age but are comparatively rare in esophageal cancers, suggesting NOTCH1 mutations drive clonal expansion but impede carcinogenesis. Here we test this hypothesis. Sequencing NOTCH1 mutant clones in aging human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygous Notch1 mutation confers a competitive advantage over wild-type cells, an effect enhanced by loss of the second allele. Widespread Notch1 loss alters transcription but has minimal effects on the epithelial structure and cell dynamics. In a carcinogenesis model, Notch1 mutations were less prevalent in tumors than normal epithelium. Deletion of Notch1 reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. Notch1 null tumors showed reduced proliferation. We conclude that Notch1 mutations in normal epithelium are beneficial as wild-type Notch1 favors tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.

Dry eye disease in mice activates adaptive corneal epithelial regeneration distinct from constitutive renewal in homeostasis

Abstract: Significance We resolve cell type-specific transcriptional signatures of corneal epithelial differentiation in health, injury, and disease; identify unique genes and gene networks that may be amenable to therapy for the treatment of ocular surface diseases; and create a framework for future studies of corneal limbal stem cells and other stem cell compartments.

Zeb1 facilitates corneal epithelial wound healing by maintaining corneal epithelial cell viability and mobility

Abstract: The cornea is the outmost ocular tissue and plays an important role in protecting the eye from environmental insults. Corneal epithelial wounding provokes pain and fear and contributes to the most ocular trauma emergency assessments worldwide. ZEB1 is an essential transcription factor in development; but its roles in adult tissues are not clear. We identify Zeb1 is an intrinsic factor that facilitates corneal epithelial wound healing. In this study, we demonstrate that monoallelic deletion of Zeb1 significantly expedites corneal cell death and inhibits corneal epithelial EMT-related cell migration upon an epithelial debridement. We provide evidence that Zeb1-regulation of corneal epithelial wound healing is through the repression of genes required for Tnfa-induced epithelial cell death and the induction of genes beneficial for epithelial cell migration. We suggest utilizing TNF-α antagonists would reduce TNF/TNFR1-induced cell death in the corneal epithelium and inflammation in the corneal stroma to help corneal wound healing.

Inflammatory and deleterious role of gut microbiota-derived trimethylamine on colon cells

Abstract: Trimethylamine (TMA) is produced by the intestinal microbiota as a by-product of metabolism of dietary precursors. TMA has been implicated in various chronic health conditions. However, the effect of TMA in the colon and the underlying mechanism was not clear. In this study, TMA exhibited toxic effects in vitro as well as in vivo. TMA-induced oxidative stress causes DNA damage, and compromised cell membrane integrity leading to the release of LDH outside the cells which ultimately leads to cell death. Besides, TMA also exhibited pronounced increase in cell cycle arrest at G2/M phase in both HCT116 and HT29 cell lines. TMA was found to be genotoxic and cytotoxic as the TMA concentration increased from 0.15 mM. A decreased ATP intracellular content was observed after 24 h, 48 h, and 72 h treatment in a time and dose-dependent manner. For in vivo research, TMA (100 mM, i.p. and intra-rectal) once a week for 12 weeks caused significant changes in cellular morphology of colon and rectum epithelium as assessed by H & E staining. TMA also significantly increased the infiltration of inflammatory cells in the colon and rectal epithelium indicating the severity of inflammation. In addition, TMA caused extensive mucosal damage and distortion in the epithelium, decrease in length of small intestine compared to control mice. In conclusion, these results highlight the detrimental effects of TMA in the colon and rectal epithelium.

Tubuloid culture enables long-term expansion of functional human kidney tubule epithelium from iPSC-derived organoids

Abstract: Significance The kidneys have highly complex architectures and functions. Advanced models are therefore required to push research into human kidney development, function, disease, and regeneration forward. Organoids from induced pluripotent stem cells and tubuloids grown from adult stem/progenitor cells are two novel models that have proven valuable for these purposes. These models each have their specific advantages and uses. Here, both protocols are combined to generate ‘iPSCod tubuloids’, a different laboratory model of the kidney with complementary advantages to both organoids and tubuloids. iPSCod tubuloids are easily handled and display key kidney functions such as waste excretion and salt reabsorption. We expect that this approach will prove useful for future research into human kidney function and disease.

Spread of premalignant mutant clones and cancer initiation in multilayered tissue

Abstract: Over 80% of human cancers originate from the epithelium, which covers the outer and inner surfaces of organs and blood vessels. In stratified epithelium, the bottom layers are occupied by stem and stem-like cells that continually divide and replenish the upper layers. In this work, we study the spread of premalignant mutant clones and cancer initiation in stratified epithelium using the biased voter model on stacked two-dimensional lattices. Our main result is an estimate of the propagation speed of a premalignant mutant clone, which is asymptotically precise in the cancer-relevant weak-selection limit. We use our main result to study cancer initiation under a two-step mutational model of cancer, which includes computing the distributions of the time of cancer initiation and the size of the premalignant clone giving rise to cancer. Our work quantifies the effect of epithelial tissue thickness on the process of carcinogenesis, thereby contributing to an emerging understanding of the spatial evolutionary dynamics of cancer.

Inflammatory Cytokine-Induced HIF-1 Activation Promotes Epithelial–Mesenchymal Transition in Endometrial Epithelial Cells

Abstract: The endometrium undergoes repeated proliferation and shedding during the menstrual cycle. Significant changes to this environment include fluctuations in the partial pressure of oxygen, exposure to a high-cytokine environment associated with intrauterine infection, and inflammation. Chronic endometritis is a condition wherein mild inflammation persists in the endometrium and is one of the causes of implantation failure and miscarriage in early pregnancy. It is thought that the invasion of embryos into the endometrium requires epithelial–mesenchymal transition (EMT)-associated changes in the endometrial epithelium. However, the effects of inflammation on the endometrium remain poorly understood. In this study, we investigated the effects of the intrauterine oxygen environment, hypoxia-inducible factor (HIF), and inflammation on the differentiation and function of endometrial epithelial cells. We elucidated the ways in which inflammatory cytokines affect HIF activity and EMT in an immortalized cell line (EM-E6/E7/TERT) derived from endometrial epithelium. Pro-inflammatory cytokines caused significant accumulation of HIF-1α protein, increased HIF-1α mRNA levels, and enhanced hypoxia-induced accumulation of HIF-1α protein. The combined effect of inflammatory cytokines and hypoxia increased the expression of EMT-inducing factors and upregulated cell migration. Our findings indicate that pro-inflammatory factors, including cytokines and LPS, work synergistically with hypoxia to activate HIF-1 and promote EMT in endometrial epithelial cells.

Derivation and characterization of new cell line from intestine of turbot (Scophthalmus maximus)

Abstract: A continuous intestine cell line from turbot (Scophthalmus maximus) designated as SMI was established utilizing the tissue explant technique. Primary SMI cell was cultured at 24 °C in a medium with 20% fetal bovine serum (FBS), then subcultured in 10% FBS after 10 passages. Impacts of medium or temperature on the growth of SMI were examined and the results indicated it grew well in DMEM supplemented with 10% FBS at 24 °C. The SMI cell line was subcultured more than 60 times. Karyotyping, chromosome number, and ribosomal RNA genotyping analysis revealed that SMI had a modal diploid chromosome number of 44 and originated from turbot. After being transfected with pEGFP-N1 and FAM-siRNA, a large number of green fluorescence signals were observed in SMI, indicating that SMI could be used as an ideal platform to explore gene function in vitro. In addition, the expression of epithelium-associated genes such as itga6, itgb4, gja1, claudin1, zo-1, and E-cadherin in SMI suggested the SMI had some characteristics of epidermal cells. The upregulation of immune-associated genes such as TNF-β, NF-κB, and IL-1β in SMI after stimulation with pathogen-associated molecular patterns suggested the SMI might exhibit immune functions similar to the intestinal epithelium in vivo.

Intraluminal neutrophils limit epithelium damage by reducing pathogen assault on intestinal epithelial cells during Salmonella gut infection

Abstract: Recruitment of neutrophils into the gut epithelium is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella Typhimurium (S. Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pre-treated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population such that the pathogen density remained high near the epithelial surface throughout the infection. The removal of luminal S. Tm by gentamicin, an antibiotic restricted to the gut lumen, reversed the effect of neutrophil depletion on epithelial cell loss. Strikingly, when using germ-free mice and an S. Tm ssaV mutant capable of epithelium invasion, but attenuated for survival and growth within host tissues, neutrophil depletion caused exacerbated immune activation of the gut mucosa and a complete destruction of the epithelial barrier. Together, our data indicate that intraluminal neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical window of the infection. Highlights ○ After the first wave of mucosal invasion (day 1 p.i.), S. Tm maintains the assault from the lumen, triggering the continued expulsion of epithelial cells in antibiotic pre-treated mice. ○ Neutrophil recruitment into the gut lumen is essential to limit this continued Salmonella attack on the epithelium. ○ In antibiotic pre-treated SPF mice, neutrophil depletion exacerbates S. Tm invasion, causing excessive epithelial cell loss, which compromises epithelial barrier integrity at later time points (day 2-3 p.i.). ○ In germ-free mice, neutrophil depletion exacerbates epithelial responses and epithelial barrier destruction even more strongly than in streptomycin pre-treated SPF mice. ○ Gentamicin treatment and ssaV mutant infections indicate that neutrophils prevent epithelial damage by eliminating and physically blocking gut-luminal pathogens.

A bioprinted 3D gut model with crypt-villus structures to mimic the intestinal epithelial-stromal microenvironment.

Abstract: The intestine is a complex tissue with a characteristic three-dimensional (3D) crypt-villus architecture, which plays a key role in the intestinal function. This function is also regulated by the intestinal stroma that actively supports the intestinal epithelium, maintaining the homeostasis of the tissue. Efforts to account for the 3D complex structure of the intestinal tissue have been focused mainly in mimicking the epithelial barrier, while solutions to include the stromal compartment are scarce and unpractical to be used in routine experiments. Here we demonstrate that by employing an optimized bioink formulation and the suitable printing parameters it is possible to produce fibroblast-laden crypt-villus structures by means of digital light projection stereolithography (DLP-SLA). This process provides excellent cell viability, accurate spatial resolution, and high printing throughput, resulting in a robust biofabrication approach that yields functional gut mucosa tissues compatible with conventional testing techniques.

A novel tool for the unbiased characterization of epithelial monolayer development in culture

Abstract: The function of an epithelial tissue is intertwined with its architecture. Epithelial tissues are often described as pseudo-two-dimensional, but this view may be partly attributed to experimental bias: many model epithelia, including cultured cell lines, are easiest to image from the "top-down." We measured the three-dimensional architecture of epithelial cells in culture and found that it varies dramatically across cultured regions, presenting a challenge for reproducibility and cross-study comparisons. We therefore developed a novel tool (Automated Layer Analysis, "ALAn") to characterize architecture in an unbiased manner. Using ALAn, we find that cultured epithelial cells can organize into four distinct architectures and that architecture correlates with cell density. Cells exhibit distinct biological properties in each architecture. Organization in the apical-basal axis is determined early in monolayer development by substrate availability, while disorganization in the apical-basal axis arises from an inability to form substrate connections. Our work highlights the need to carefully control for three-dimensional architecture when using cell culture as a model system for epithelial cell biology and introduces a novel tool, built on a set of rules that can be widely applied to epithelial cell culture.

Inflammation Confers Healing Advantage to Corneal Epithelium Following Subsequent Injury

Abstract: Recent evidence shows that epithelial stem/progenitor cells in barrier tissues such as the skin, airways and intestines retain a memory of previous injuries, which enables tissues to accelerate barrier restoration after subsequent injuries. The corneal epithelium, the outermost layer of the cornea, is the frontline barrier for the eye and is maintained by epithelial stem/progenitor cells in the limbus. Herein, we provide evidence that inflammatory memory also exists in the cornea. In mice, eyes that had been exposed to corneal epithelial injury exhibited faster re-epithelialization of the cornea and lower levels of inflammatory cytokines following subsequent injury (either the same or a different type of injury) relative to naïve eyes without previous injury. In ocular Sjögren’s syndrome patients, corneal punctate epithelial erosions were significantly reduced after experiencing infectious injury compared with before. These results demonstrate that previous exposure of the corneal epithelium to inflammatory stimuli enhances corneal wound healing in response to a secondary assault, a phenomenon which points to the presence of nonspecific inflammatory memory in the cornea.

Histological Changes Indused by Monosodium Glutamate on the Growth and Development of the Offsprings Albino Mice Cornea

Abstract: This research aim to study the effect of different doses of a Monosodium Glutamate (MSG) to cause histopathological changes in the offspring corneal for the ages 7, 15, 21, 30, and 60 days. The mothers, type Mus musculus mice has been orally administrated from 17th day of pregnancy by different concentrations (2, 4, and 8 g/Kg/ body weight) of MSG using stomach tube. Microscopic examination showed no changes in the cornea for a seventh day and administrated concentrations of 2 and 4 g / kg body weight, but observed degeneration of fibre stroma, necrosis and the reduction of the anterior epithelial layer of the cornea at dose of 8 g / kg. In the group of 15 days, these changes are mentioned; hyperplasia in the anterior epithelium of the peripheral cornea of 2 g, deformation in the peripheral part of the cornea and the reduction of thickness and intensify the stroma components at a concentration of 4 g either at a concentration of 8 g / kg has appeared hyperplasia in fibroblast cells in the corneal stroma and wave of the basement membrane of anterior epithelial tissue of central cornea. In a group 21days, these changes are shown; necrosis and rarefaction in stroma of central cornea while the reduction of the anterior epithelial layer of the peripheral cornea notes and wave anterior surface of the cornea in the concentration of 2 g / kg. Deformation appeared and reduction layers and irregular with dissociation in the stromal fibers in some areas in the concentration of 4 g / kg, while in the concentration of 8 g / kg show damage in the anterior part of epithelial of the central corneal tissue. In a group 30 days and concentration of 2 g / kg, these changes were observed; necrosis, swelling in some anterior epithelial cells, condensation of nuclei in endothelial cells, with Keratinized at the surface of the cornea and edema in the stroma. Bulge appeared in the stroma of peripheral cornea and hyperplasia in fiberoblast at a concentration of 4g/kg, meanwhile at a concentration of 8 g/kg show necrosis under the anterior epithelium of central cornea and dissociation of stromal fiberoblast. In the group 60 Days at the concentration of 2 g / kg show damage in the anterior epithelial tissue of the peripheral cornea and hyperplasia in some parts of the anterior epithelial tissue in the central cornea. At the concentration of 4g/kg show damage in the stromal fibers of peripheral corneal epithelium and detachment of endothelial tissue. In concentration 8 g/kg reduction appeared in the anterior epithelial tissue in the certain part of the cornea, hyperplasia in endothelial tissue, condensed layer appeared on the surface of anterior epithelial tissue.

Inulin diet uncovers complex diet-microbiota-immune cell interactions remodeling the gut epithelium

Abstract: The continuous proliferation of intestinal stem cells followed by their tightly regulated differentiation to epithelial cells is essential for the maintenance of the gut epithelial barrier and its functions. How these processes are tuned by diet and gut microbiome is an important, but poorly understood question. Dietary soluble fibers, such as inulin, are known for their ability to impact the gut bacterial community and gut epithelium, and their consumption has been usually associated with health improvement in mice and humans. In this study, we tested the hypothesis that inulin consumption modifies the composition of colonic bacteria and this impacts intestinal stem cells functions, thus affecting the epithelial structure.Mice were fed with a diet containing 5% of the insoluble fiber cellulose or the same diet enriched with an additional 10% of inulin. Using a combination of histochemistry, host cell transcriptomics, 16S microbiome analysis, germ-free, gnotobiotic, and genetically modified mouse models, we analyzed the impact of inulin intake on the colonic epithelium, intestinal bacteria, and the local immune compartment.We show that the consumption of inulin diet alters the colon epithelium by increasing the proliferation of intestinal stem cells, leading to deeper crypts and longer colons. This effect was dependent on the inulin-altered gut microbiota, as no modulations were observed in animals deprived of microbiota, nor in mice fed cellulose-enriched diets. We also describe the pivotal role of γδ T lymphocytes and IL-22 in this microenvironment, as the inulin diet failed to induce epithelium remodeling in mice lacking this T cell population or cytokine, highlighting their importance in the diet-microbiota-epithelium-immune system crosstalk.This study indicates that the intake of inulin affects the activity of intestinal stem cells and drives a homeostatic remodeling of the colon epithelium, an effect that requires the gut microbiota, γδ T cells, and the presence of IL-22. Our study indicates complex cross kingdom and cross cell type interactions involved in the adaptation of the colon epithelium to the luminal environment in steady state. Video Abstract.

Foregut ultrastructure of adult sanguinicola volgensis (rašín, 1929) mcintosh, 1934 (digenea: aporocotylidae).

Abstract: Herein, we use scanning and transmission electron microscopy to describe the foregut (mouth, pharyngeal canal, and associated epithelia and musculature) of an adult freshwater fish blood fluke, Sanguinicola volgensis (Rašín, 1929) McIntosh, 1934, infecting the blood of sabre, Pelecus cultratus Linnaeus, 1758 (Cypriniformes: Leuciscidae) from the Volga River, Russia. Our results indicate that S. volgensis has a pharynx and lacks an oral sucker and that its pharyngeal canal acts as a peristaltic pump that sucks blood into the esophagus, whereupon digestion commences with granules secreted from the esophageal epithelium. We saw no evidence of longitudinal muscle fibers beneath the pharyngeal canal epithelium, pharyngeal glands, or pharyngeal epithelial cells or muscle cells within the pharyngeal muscular complex; collectively indicating the presence of a pharynx rather than an oral sucker. The specialized epithelial lining associated with the mouth and pharyngeal canal evidently is unique among neodermatans; it is smooth, ∼40 nm thick anteriorly, and thickens (∼250-700 nm) posteriorly as the mouth cavity transitions into the pharyngeal canal. The pharyngeal canal epithelium has lumps of dense material resembling those of the basal lamina and fibrous coat of the tegument. The actin-like material within the pharyngeal cavity epithelium could provide structural support to the pharynx.

Neutrophilic inflammation promotes SARS-CoV-2 infectivity and augments the inflammatory responses in airway epithelial cells

Abstract: Introduction In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre- existing co-morbidities correlating to incidence to severe COVID-19 are associated with chronic airway neutrophilia. Furthermore, examination of COVID-19 explanted lung tissue revealed a series of epithelial pathologies associated with the infiltration and activation of neutrophils, indicating neutrophil activity in response to SARS-CoV-2 infection. Methods To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory responses to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. This model was infected with live SARS-CoV-2 virus the epithelial response to infection was evaluated. Results SARS-CoV-2 infection of airway epithelium alone does not result in a notable pro-inflammatory response from the epithelium. The addition of neutrophils induces the release of proinflammatory cytokines and stimulates a significantly augmented proinflammatory response subsequent SARS-CoV-2 infection. The resulting inflammatory responses are polarized with differential release from the apical and basolateral side of the epithelium. Additionally, the integrity of the \epithelial barrier is impaired with notable epithelial damage and infection of basal stem cells. Conclusions This study reveals a key role for neutrophil-epithelial interactions in determining inflammation and infectivity.

Spatio-temporal remodeling of extracellular matrix orients epithelial sheet folding

Abstract: Biological systems are inherently noisy; however, they produce highly stereotyped tissue morphology. Drosophila pupal wings show a highly stereotypic folding through uniform expansion and subsequent buckling of wing epithelium within a surrounding cuticle sac. The folding pattern produced by buckling is generally stochastic; it is thus unclear how buckling leads to stereotypic tissue folding of the wings. We found that the extracellular matrix (ECM) protein, Dumpy, guides the position and direction of buckling-induced folds. Dumpy anchors the wing epithelium to the overlying cuticle at specific tissue positions. Tissue-wide alterations of Dumpy deposition and degradation yielded different buckling patterns. In summary, we propose that spatio-temporal ECM remodeling shapes stereotyped tissue folding through dynamic interactions between the epithelium and its external structures. One-Sentence Summary Remodeling of extracellular matrix protein, Dumpy, guides epithelial tissue folding during Drosophila wing development.

Effect of increased intake of concentrates and sodium butyrate supplementation on ruminal epithelium structure and function in growing rams

Abstract: Increased ruminal butyrate production is considered to have a positive impact on rumen epithelium growth and function. However, excessive ruminal butyrate production may affect the rumen negatively, particularly when rumen is already challenged with low pH. The aim of this study was to determine the effect of the inclusion of concentrates in the diet and sodium butyrate (SB) supplementation on ruminal epithelium growth and function in growing rams. Forty-two rams (27.8 ± 7.3 kg; 9 to 14 months of age) were allocated into six treatments and fed a diet with low (22.5% of diet DM; LOW) or high (60% of diet DM; HIGH) inclusion of concentrates in combination with no (SB0), 1.6% (SB1.6) or 3.2% of diet DM (SB3.2) inclusion of SB. There was no impact of the investigated factors on papilla dimensions and mucosa surface area, either in the atrium ruminis or ventral rumen (P ≥ 0.11). Stratum corneum thickness was higher for HIGH compared to LOW treatments (P ≤ 0.04), independently of the location in the rumen. In the atrium ruminis, the epithelium and living strata thickness quadratically increased due to SB supplementation for LOW treatments but quadratically decreased for HIGH treatments (concentrate inclusion × butyrate supplementation interaction; P ≤ 0.03); conversely, in the ventral sac of the rumen, a thicker epithelium was observed due to both increased concentrates inclusion in the diet and SB supplementation (P < 0.01) but living strata thickness was increased only by SB supplementation (linear effect; P < 0.01). The epithelium damage index in the ventral sac of the rumen was higher for LOW compared to HIGH treatments (P = 0.02). Increased concentrates inclusion in the diet increased mRNA expression of monocarboxylate transporter 1 in both the epithelium of the atrium ruminis and ventral rumen, occludin in the epithelium of the atrium ruminis and downregulated in adenoma in the epithelium of the ventral rumen (P ≤ 0.02). Protein expression of claudin-4 in the epithelium of the ventral rumen was the highest for the HIGH/SB1.6 and HIGH/SB3.2 treatments (significant effect of interaction between main effects; P < 0.01). Under the conditions of the current study, increased intake of concentrates had mostly positive effects on ruminal epithelium in growing rams, and the same was observed for the effect of SB supplementation. However, the effect of SB supplementation was at least partially affected by the inclusion of concentrates in the diet.

MLN4924 Promotes Self-Renewal of Limbal Stem Cells and Ocular Surface Restoration

Abstract: Objective: To study the role of MLN4924 in corneal stem cell maintenance and corneal injury repair. Methods: In cell experiments, the Sprague–Dawley (SD) rat corneal epithelial cells were co-cultured with mitomycin C-inactivated mouse feeder cells in a supplemental hormonal epithelial medium (SHEM) with or without MLN4924. Cells were photographed using an optical microscope. Furthermore, we performed crystal violet, polymerase chain reaction (PCR), and immunofluorescence staining on limbal stem cells (LSCs). In animal experiments, we scraped the corneal epithelium with a central corneal diameter of 4 mm in SD rats. The area of the corneal epithelial defect was calculated by fluorescein sodium staining. Results: LSCs in the MLN4924 group had significantly proliferated. The MLN4924 treatment evidently enhanced the clone formation rate and clone area of LSCs. The expression levels of Ki67, p63, ABCG2, Bmi1, and C/EBPδ increased in LSCs after MLN4924 treatment, whereas the expression of K12 decreased. At 12 and 24 h after scraping, the corneal epithelium recovery rate in the eyes of the MLN4924-treated rats was accelerated. Conclusions: MLN4924 can maintain stemness, reduce differentiation, promote the proliferative capacity of rat LSCs, and accelerate corneal epithelial wound healing in SD rats.

New insight into Epstein-Barr virus infection using models of stratified epithelium

Abstract: Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is transmitted in saliva. EBV transits through the oral epithelium to infect B cells, where it establishes a life-long latent infection. Reinfection of the epithelium is believed to be mediated by virus shed from B cells, but whether a latent reservoir can exist in the epithelia is unknown. We previously developed an in vitro organotypic model of stratified epithelium where EBV can readily replicate within the suprabasal layers of the epithelium following apical infection mediated by virus-producing B cells. Given that infected epithelial cells and cell-free virus are observed in saliva, we examined the ability of both of these to mediate infection in organotypic cultures. Epithelial-derived cell-free virus was able to infect organotypic cultures from the apical surface, but showed enhanced infection of B cells. Conversely, B cell-derived virus exhibited enhanced infection of epithelial cells. While EBV has been detected in basal cells in oral hairy leukoplakia, it is unknown whether EBV can be seen in undifferentiated primary keratinocytes in the basal layer. Undifferentiated epithelial cells expressed proposed EBV receptors in monolayer and were susceptible to viral binding and entry. Integrins, and occasionally ephrin A2, were expressed in the basal layer of gingiva and tonsil derived organotypic cultures, but the known B-cell receptors HLAII and CD21 were not detected. Following infection with cell-free virus or virus-producing B cells at either the apical or basolateral surface of preformed organotypic cultures, abundant infection was detected in differentiated suprabasal cells while more limited but readily detectable infection was observed in the undifferentiated basal cells. Together, our data has provided new insight into EBV infection in stratified epithelium.

Effect of PKH-26-labeled exosomes derived from bone marrow mesenchymal stem cells on corneal epithelium regeneration in diabetic mice

Abstract: Background It is known that bone marrow mesenchymal stem cells (BM-MSCs) could speed up the regeneration of diabetic corneal epithelium. To investigate the effect of exosomes derived from mouse BM-MSCs on corneal epithelium regeneration in diabetic mice. Methods Diabetic mouse models were established using streptozotocin (STZ), and their central corneal epithelium was scratched under a microscope. The diabetic mice were randomly divided into three groups: the control group was injected with subconjunctival phosphate buffer saline; the exosomes group was treated with a subconjunctival injection of exosomes derived from BM-MSCs; and the BM-MSCs group was treated with a subconjunctival injection of BM-MSCs. The corneal epithelium repair rates in the three groups were compared, and the distribution of the exosomes derived from BM-MSCs labeled with PKH-26 was observed by immunofluorescence. Hematoxylin-eosin staining of the corneal tissue was observed 72 h after the treatments in the three groups. Results The diabetic mice were successfully established by a blood glucose level >16.7 mmol/L after 8 weeks. The corneal epithelium healing rates in experimental groups 1 and 2 were significantly higher than those of the control group at 24, 48, and 72 h (P<0.05). However, there was no significant difference in the corneal epithelial healing rate between experimental groups 1 and 2 (P>0.05). The exosomes derived from BM-MSCs were found in the superficial corneal stroma in experimental groups 1 and 2, with the majority of the exosomes distributed in the limbal epithelium at the edge of the injury area. The proliferation of corneal epithelial cells in experimental groups 1 and 2 was more obvious than that in the control group. Conclusions The exosomes derived from BM-MSCs labeled with PKH-26 significantly promoted the repair of corneal epithelial injury in diabetic mice. These exosomes might be a substitute for BM-MSCs in the repair of diabetic keratopathy, suggesting a new idea for the repair of diabetic keratopathy with “cell-free” stem cell therapy, which will require a clinical study.