SHED-derived exosomes ameliorate hyposalivation caused by Sjögren's syndrome via Akt/GSK-3β/Slug-mediated ZO-1 expression / 中华医学杂志(英文版)

BACKGROUND@#Sjögren's syndrome (SS) is an autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The treatment is still challenging. This study aimed to explore the therapeutic role and mechanism of exosomes obtained from the supernatant of stem cells derived from human exfoliated deciduous teeth (SHED-exos) in sialadenitis caused by SS.@*METHODS@#SHED-exos were administered to the submandibular glands (SMGs) of 14-week-old non-obese diabetic (NOD) mice, an animal model of the clinical phase of SS, by local injection or intraductal infusion. The saliva flow rate was measured after pilocarpine intraperitoneal injection in 21-week-old NOD mice. Protein expression was examined by western blot analysis. Exosomal microRNA (miRNAs) were identified by microarray analysis. Paracellular permeability was evaluated by transepithelial electrical resistance measurement.@*RESULTS@#SHED-exos were injected into the SMG of NOD mice and increased saliva secretion. The injected SHED-exos were taken up by glandular epithelial cells, and further increased paracellular permeability mediated by zonula occluden-1 (ZO-1). A total of 180 exosomal miRNAs were identified from SHED-exos, and Kyoto Encyclopedia of Genes and Genomes analysis suggested that the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway might play an important role. SHED-exos treatment down-regulated phospho-Akt (p-Akt)/Akt, phospho-glycogen synthase kinase 3β (p-GSK-3β)/GSK-3β, and Slug expressions and up-regulated ZO-1 expression in SMGs and SMG-C6 cells. Both the increased ZO-1 expression and paracellular permeability induced by SHED-exos were abolished by insulin-like growth factor 1, a PI3K agonist. Slug bound to the ZO-1 promoter and suppressed its expression. For safer and more effective clinical application, SHED-exos were intraductally infused into the SMGs of NOD mice, and saliva secretion was increased and accompanied by decreased levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and Slug and increased ZO-1 expression.@*CONCLUSION@#Local application of SHED-exos in SMGs can ameliorate Sjögren syndrome-induced hyposalivation by increasing the paracellular permeability of glandular epithelial cells through Akt/GSK-3β/Slug pathway-mediated ZO-1 expression.

Fibroblasts overpressing WNT2b cause impairment of intestinal mucosal barrier / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD).@*METHODS@#Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins.@*RESULTS@#In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa.@*CONCLUSION@#Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.

Caracterización de las causas de alopecia infantil / Aetiology of childhood alopecia

Introducción: La alopecia infantil es una afección poco frecuente en la consulta dermatológica pediátrica. Su etiología es variable según el grupo etario estudiado. El objetivo fue estudiar la causa de alopecia en niños en 2 hospitales pediátricos de referencia nacional en Chile. Pacientes y método: Análisis descriptivo de registros clínicos del total de pacientes atendidos entre enero de 2007 y junio de 2010 en los Servicios de Dermatología de los Hospitales Roberto del Río y Luis Calvo Mackenna. Se incluyeron pacientes con diagnóstico clínico de alopecia. Resultados: Se encontraron 345 registros clínicos, 179 varones (51,9%). La mediana de edad fue 72 meses. Los diagnósticos más prevalentes fueron alopecia areata (AA) (36,8%), tiña capitis (TC) (21%), nevo sebáceo (13,2%) y efluvio telógeno (8,7%). Según el grupo etario predominaron en recién nacidos: aplasia cutis y nevo sebáceo; en lactantes, preescolares y escolares: nevo sebáceo, AA y TC. En escolares se agregó tricotilomanía. En adolescentes nevo sebáceo, AA y efluvio telógeno. Se observó una correlación significativa entre AA con enfermedad autoinmune, enfermedad tiroidea, alteraciones ungueales, enfermedad psiquiátrica y síndrome de Down. En TC el agente etiológico más prevalente fue Microsporum Canis (86,6%). La tricotilomanía se correlacionó con enfermedad psiquiátrica significativamente. Conclusiones: Las principales causas de alopecia infantil fueron adquiridas y no cicatriciales. La etiología varía de acuerdo al grupo etario estudiado. Algunos tipos de alopecia infantil presentaron alta prevalencia de enfermedad psiquiátrica.

Introduction: Childhood alopecia is a relative rare event in general paediatric dermatology practice. Hair loss in children may have multiple causes, and there are different types of alopecia according to age groups. The aim of the study was to describe the clinical and epidemiological profile of alopecia in children from two Chilean paediatric hospitals. Patients and method: Descriptive analysis of clinical records of patients from the Dermatology Department of Roberto del Rio and Luis Calvo Mackenna Hospitals between January 2007 and June 2010. Patients with clinical diagnosis of alopecia were included. Results: A total of 345 clinical records were analysed, with 179 males (51.9%). The median age was 72 months. Overall, the most common diagnoses were: alopecia areata (AA), (36.8%), tinea capitis (TC), (21%), nevus sebaceous (13.2%), and tellogen effluvium (8.7%). According to age groups, in newborns, the most common causes were aplasia cutis and nevus sebaceous. In toddlers, pre-school and school children, the principal causes were nevus sebaceous, AA and TC. Trichotillomania was also significant in school children. In adolescents, nevus sebaceous, AA and tellogen effluvium were the most frequent diagnoses. AA was statistically associated with autoimmune disease, thyroid disease, nail disorder, psychiatric disease, and Down's syndrome. The most common aetiological agent in TC was M. canis (86.6%). Trichotillomania was also statistically associated to psychiatric disorders. Conclusions: In this study, the main causes of alopecia in children were acquired and non-scarring alopecia. In our results, the type of alopecia varies according to age group. Some types of childhood alopecia showed a close correlation to psychiatric disorders.

Expression of Zonula Occludens-1 in Cerebral Cortex Following Traumatic Brain Injury / 法医学杂志

OBJECTIVE@#To observe the time-course expression of zonula occludens-1 (ZO-1) in cerebral cortex after traumatic brain injury (TBI).@*METHODS@#The TBI model of mouse was established. The mice were divided in 1 h, 3 h, 6 h, 12 h, 24 h, 3 d, 7 d after TBI, sham and control groups. The permeability of the blood brain barrier was evaluated by measuring the extravasation of Evans blue (EB) dye. The expression of ZO-1 in cerebral cortex in the injured area was detected by Western blotting and immunohistochemistry.@*RESULTS@#The extravasation of EB dye of injured cortex gradually increased from 1 h, peaked at 1-3 d and approximately decreased to normal at 7 d after TBI. Western blotting revealed that the expression of ZO-1 gradually decreased after 1 h, was at the lowest at 1-3 d, and then significantly increased after 7 d but was still lower than that of normal and sham groups. The result of immunohistochemistry showed that ZO-1 had strong expression in vessel of normal cortex, gradually decreased after TBI, and almost disappeared at 3 d after TBI and gradually recovered to normal level later.@*CONCLUSION@#The expression of ZO-1 in the injured cortex after TBI initially decreases and then increases. The negative correlation between ZO-1 expression and EB extravasation after TBI could be used as a new indicator for wound age estimation.

Hypoxia Increases Epithelial Permeability in Human Nasal Epithelia

PURPOSE: The nasal mucosa is the first site to encounter pathogens, and it forms continuous barriers to various stimuli. This barrier function is very important in the innate defense mechanism. Additionally, inflammation of the nasal sinus is known to be a hypoxic condition. Here, we studied the effect of hypoxia on barrier function in normal human nasal epithelial (NHNE) cells. MATERIALS AND METHODS: The expression levels of various junction complex proteins were assessed in hypoxia-stimulated NHNE cells and human nasal mucosal tissues. We performed real-time polymerase chain reaction analysis, western blotting, and immunofluorescence assays to examine differences in the mRNA and protein expression of ZO-1, a tight junction protein, and E-cadherin in NHNE cells. Moreover, we evaluated the trans-epithelial resistance (TER) of NHNE cells under hypoxic conditions to check for changes in permeability. The expression of ZO-1 and E-cadherin was measured in human nasal mucosa samples by western blotting. RESULTS: Hypoxia time-dependently decreased the expression of ZO-1 and E-cadherin at the gene and protein levels. In addition, hypoxia decreased the TER of NHNE cells, which indicates increased permeability. Human nasal mucosa samples, which are supposed to be hypoxic, showed significantly decreased levels of ZO-1 and E-cadherin expression compared with control. CONCLUSION: Our results demonstrate that hypoxia altered the expression of junction complex molecules and increased epithelial permeability in human nasal epithelia. This suggests that hypoxia causes barrier dysfunction. Furthermore, it may be associated with innate immune dysfunction after encountering pathogens.

Acute Modulations in Stratum Corneum Permeability Barrier Function Affect Claudin Expression and Epidermal Tight Junction Function via Changes of Epidermal Calcium Gradient

Tight junction (TJ) is recognized as a second barrier of the skin. Altered expression of TJ proteins in various skin diseases characterized by the abnormal permeability barrier such as psoriasis suggests that TJ could be affected by stratum corneum (SC) barrier status. However, the physiological relationship between SC and TJ barrier remains to be investigated. Therefore, we examined the effect of SC barrier disruption on the expression of TJ proteins, claudin (Cldn)-1 and Cldn-4, and TJ barrier function in hairless mouse skin. We also investigated whether the alterations in epidermal Ca2+ affected TJ proteins expression in vivo. Repeated tape-stripping induced a sequential change of the expression and function of TJ. As early as 15-30 minutes after tape-stripping, downregulation of Cldn-1 and Cldn-4 immunoreactivity and protein level without change in mRNA level was found. This was accompanied by the abnormal leakage of lanthanum. However, by 1 hour Cldn-1 and Cldn-4 immunolocalization recovered along with normalized lanthanum permeation pattern. Moreover, the mRNA and protein levels of Cldn-1 and Cldn-4 were increased by 1 to 6 hours after tape-stripping. Inhibition of calcium loss by immersion of barrier-disrupted skin into a high Ca2+ solution prevented the dislocation of Cldn-1 and Cldn-4. Occlusion of barrier-disrupted skin delayed the restoration of Cldn-1 and Cldn-4. Our results suggest that the alteration of epidermal Ca2+ gradient caused by SC barrier perturbation affects the TJ structure and function and the faster recovery of TJ as compared to the SC barrier may imply the protective homeostatic mechanism of skin barrier.

Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-á) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-á (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-á treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-á decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-á did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-á increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.