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tight junction [keywords]
- Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line. [Journal Article]
- PLoS One 2014; 9(10):e110916.
The mucosal surface of intestine is continuously exposed to both potential pathogens and beneficial commensal microorganisms. Recent findings suggest that intestinal epithelial cells, which once considered as a simple physical barrier, are a crucial cell lineage necessary for maintaining intestinal immune homeostasis. Therefore, establishing a stable and reliable intestinal epithelial cell line for future research on the mucosal immune system is necessary. In the present study, we established a porcine intestinal epithelial cell line (ZYM-SIEC02) by introducing the human telomerase reverse transcriptase (hTERT) gene into small intestinal epithelial cells derived from a neonatal, unsuckled piglet. Morphological analysis revealed a homogeneous cobblestone-like morphology of the epithelial cell sheets. Ultrastructural indicated the presence of microvilli, tight junctions, and a glandular configuration typical of the small intestine. Furthermore, ZYM-SIEC02 cells expressed epithelial cell-specific markers including cytokeratin 18, pan-cytokeratin, sucrase-isomaltase, E-cadherin and ZO-1. Immortalized ZYM-SIEC02 cells remained diploid and were not transformed. In addition, we also examined the host cell response to Salmonella and LPS and verified the enhanced expression of mRNAs encoding IL-8 and TNF-α by infection with Salmonella enterica serovars Typhimurium (S. Typhimurium). Results showed that IL-8 protein expression were upregulated following Salmonella invasion. TLR4, TLR6 and IL-6 mRNA expression were upregulated following stimulation with LPS, ZYM-SIEC02 cells were hyporeponsive to LPS with respect to IL-8 mRNA expression and secretion. TNFα mRNA levels were significantly decreased after LPS stimulation and TNF-α secretion were not detected challenged with S. Typhimurium neither nor LPS. Taken together, these findings demonstrate that ZYM-SIEC02 cells retained the morphological and functional characteristics typical of primary swine intestinal epithelial cells and thus provide a relevant in vitro model system for future studies on porcine small intestinal pathogen-host cell interactions.
- Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption. [Journal Article]
- PLoS One 2014; 9(10):e111401.
Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.
- 5-Fluorouracil-induced changes of intestinal integrity biomarkers in BALB/c mice. [Journal Article]
- J Cancer Prev 2013 Dec; 18(4):322-9.
Intestinal mucositis is a most frequently occurring toxicity in cancer chemotherapy, and consequent malnutrition reduces tolerance to cancer therapies. Therefore it is important to lessen the severity of mucotitis and to develop complementary agents capable of reducing mucotitis-related symptoms. This study was conducted to determine 5-fluorouracil (5-FU) induced intestinal damage to understand intestinal damages due to chemotherapy and to provide information on biomarkers which can be used to screen complementary agents in future studies.BALB/c mice were divided into three experimental groups and subjected to the intraperitoneal injection of either 100 mg/kg or 200 mg/kg of 5-FU. The third group was used as PBS controls. Body weights and the consistency of the stools were recorded every day, and the animals were sacrificed on the 7th day post 5-FU administration. The expressions of intestinal tight junction proteins and mRNAs of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were determined.The body weight of the animals treated with 5-FU was significantly decreased in a dose-dependent manner. However, mice given 100 mg/kg 5-FU rapidly recovered the original body weight. Symptom of diarrhea was also more severe in 200 mg/kg 5-FU treated group than that of the 100 mg/kg 5-FU treated animals. The expressions of occludin and claudin-1, not ZO-1 protein expressions in 200 mg/kg 5-FU treated animals were significantly reduced compared to those of the control group or 100 mg/kg 5-FU group. The expression of Nuclear factor-kappa B p65 (NF-κB p65) protein and TNF-α mRNA were significantly higher in 5-FU treated group compared to those of control group. No difference was observed with IL-1β expression.These results suggested that selected tight junction proteins and inflammatory cytokines are related to 5-FU induced mucositis, and thereby can be used as targets of developing complementary agents.
- Cholesterol-enriched diet disrupts the blood-testis barrier in rabbits. [JOURNAL ARTICLE]
- Am J Physiol Endocrinol Metab 2014 Oct 21.:ajpendo.00416.2014.
About 15% of heterosexual couples in the USA suffer from infertility issues; male infertility accounts for approximately 50% of all infertility cases and roughly 50% of male infertility is idiopathic. Increased levels of plasma cholesterol affect negatively spermatogenesis and male fertility, but by unclear mechanisms. Clearly, spermatogenesis occurs in immune-privileged seminiferous tubules that are protected by the blood-testis barrier (BTB) and BTB disruption results in sperm damage and male infertility. Accordingly, using rabbits fed a 2% cholesterol-enriched diet for 2, 4, and 6 weeks to raise levels of plasma cholesterol we tested the hypothesis that elevated levels of plasma cholesterol disrupt functionally and biochemically the BTB. The cholesterol-enriched diet increased dramatically and time-dependently lipid deposition in the seminiferous tubules and disrupted the BTB as evidenced by increased IgG staining within the seminiferous tubules. Total protein levels of the tight junction proteins ZO-1 and occludin were increased in the seminiferous tubules of rabbits fed the cholesterol-enriched diet and the distribution patterns of tight junction proteins were markedly affected including an increased accumulation of tight junction proteins in endosomes. Disruption of the integrity of the BTB due to increased plasma levels of cholesterol might play a role in male infertility.
- Serum zonulin is elevated in women with polycystic ovary syndrome, and correlates with insulin resistance and severity of anovulation. [JOURNAL ARTICLE]
- Eur J Endocrinol 2014 Oct 21.
Objective: Evidence suggests that increased gut permeability may be associated with polycystic ovary syndrome (PCOS). Human zonulin is currently the only physiological mediator known to regulate gut permeability reversibly by disassembling intestinal tight junctions. So far, no data on serum zonulin levels in patients with PCOS is available. This study aimed to determine circulating serum zonulin levels in women with PCOS, and discuss the relationship between zonulin, insulin resistance and menstrual disorders in this group. Design: Case-control study. Methods: Seventy-eight women recently diagnosed with PCOS, and 63 age matched healthy controls were recruited for this study. Serum zonulin levels were determined by ELISA. Insulin resistance was assessed by homeostasis model assessment of insulin resistance (HOMA-IR) and Matsuda and DeFronzo's insulin sensitivity index (ISI). Results: PCOS women had higher serum zonulin (p=0.022). After adjustment for age and body mass index, zonulin levels significantly correlated with HOMA-IR and ISI. Furthermore, PCOS women with more severe menstrual disorders had significantly higher zonulin levels and displayed an inverse correlation between zonulin and the number of menstrual cycles per year (r=-0.398, p<0.001). Conclusions: Serum zonulin, a biomarker for gut permeability, is increased in PCOS women, and correlates with insulin resistance and severity of menstrual disorders. It suggests that alterations of gut permeability may play a role in the patho-physiology of PCOS, and serum zonulin might be used as a biomarker for both risk stratification and therapeutic outcomes in PCOS women.
- Genetic ablation of afadin causes mislocalization and deformation of paneth cells in the mouse small intestinal epithelium. [Journal Article]
- PLoS One 2014; 9(10):e110549.
Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.
- Targeted 25-Hydroxyvitamin D3 1α-Hydroxylase Adoptive Gene Therapy Ameliorates DSS-induced Colitis without Causing Hypercalcemia in Mice. [JOURNAL ARTICLE]
- Mol Ther 2014 Oct 20.
Systemic 1,25(OH)2D3 treatment ameliorating murine IBD could not be applied to patients because of hypercalcemia. We tested the hypothesis that increasing 1,25(OH)2D3 synthesis locally by targeting delivery of the 1α-hydroxylase gene (CYP27B1) to the inflamed bowel would ameliorate IBD without causing hypercalcemia. Our targeting strategy is the use of CD11b(+)/Gr1(+) monocytes as the cell vehicle and a macrophage-specific promoter (Mac1) to control CYP27B1 expression. The CD11b(+)/Gr1(+) monocytes migrated initially to inflamed colon and some healthy tissues in DSS colitis mice; however, only the migration of monocytes to the inflamed colon was sustained. Adoptive transfer of Gr1(+) monocytes did not cause hepatic injury. Infusion of Mac1-CYP27B1-modified monocytes increased body weight gain, survival, and colon length, and expedited mucosal regeneration. Expression of pathogenic Th17 and Th1 cytokines (IL-17a and IFN-γ) was decreased, while expression of protective Th2 cytokines (IL-5 and IL-13) was increased, by the treatment. This therapy also enhanced tight junction gene expression in the colon. No hypercalcemia occurred following this therapy. In conclusion, we have for the first time obtained proof-of-principle evidence for a novel monocyte-based adoptive CYP27B1 gene therapy using a mouse IBD model. This strategy could be developed into a novel therapy for IBD and other autoimmune diseases.Molecular Therapy (2014); doi:10.1038/mt.2014.201.
- Matrix Metalloproteinase-3 Promotes Early Blood-Spinal Cord Barrier Disruption and Hemorrhage and Impairs Long-Term Neurological Recovery after Spinal Cord Injury. [JOURNAL ARTICLE]
- Am J Pathol 2014 Oct 1.
After spinal cord injury (SCI), blood-spinal cord barrier (BSCB) disruption by matrix metalloproteinases (MMPs) leads to BSCB permeability and blood cell infiltration, contributing to permanent neurological disability. Herein, we report that MMP-3 plays a critical role in BSCB disruption after SCI in mice. MMP-3 was induced in infiltrated neutrophils and blood vessels after SCI, and NF-κB as a transcription factor was involved in MMP-3 expression. BSCB permeability and blood cell infiltration after injury were more reduced in Mmp3 knockout (KO) mice than in wild-type (WT) mice, which was significantly inhibited by Mmp3 siRNA or a general inhibitor of MMPs, N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. The level of tight junction proteins, such as occludin and zonula occludens-1, which decreased after SCI, was also higher in Mmp3 KO than in WT mice. Exogenously, MMP-3 injection into the normal spinal cord also induced BSCB permeability. Furthermore, MMP-9 activation after injury was mediated by MMP-3 activation. Finally, improved functional recovery was observed in Mmp3 KO mice compared with WT mice after injury. These results demonstrated the role of MMP-3 in BSCB disruption after SCI for the first time and suggest that the regulation of MMP-3 can be considered a therapeutic target to inhibit BSCB disruption and hemorrhage, and thereby enhance functional recovery after acute SCI.
- The Effect of Claudin-5 Overexpression on the Interactions of Claudin-1 and -2 and Barrier Function in Retinal Cells. [JOURNAL ARTICLE]
- Curr Mol Med 2014 Oct 15.
Claudin-5, one of the dominant tight junctions (TJs) proteins, plays an important role in maintaining the barrier function in the blood brain and retinal barrier. This study aimed to investigate the effect of claudin-5 overexpression on the interactions of claudin-1 and -2 and barrier functions in primary cultured human retinal pigment epithelium cells (HRPECs) and human retina endothelial cells (HRECs). Lentivirus was used to mediate the overexpression of claudin-5 in retinal cells. Significantly increased mRNA and protein levels of claudin-5 were detected in the transfection group. After the transfected cells grew on the transwell membrane for three weeks, a stable monolayer cell barrier model was established in vitro. The claudins expressions analysis showed that overexpressed claudin-5 significantly increased the expression of claudin-1, while it decreased the expression of claudin-2 in both mRNA and protein level. Co-IP experiments and barrier function assay revealed that claudin-5 overexpression promoted the interactions of claudin-1 and claudin-2 and enhanced the barrier function of retinal cells. Intriguingly, the exogenous expression of claudin-5 induced new interaction pattern between claudin-5 and claudin-1 or -2 in HRPECs, which do not have endogenous claudin-5 expression. In addition, claudin-5 overexpression decreased cell mobility and the sprouting capability of vessel tube formation in vitro. This study demonstrated that claudin-5 has a positive regulation in the formation of retinal barrier. Claudin elements and their interactions can be modulated and that such dynamic properties are important for the functions of TJs, ranging from the regulation of retinal barrier integrity to junction-associated signaling mechanisms.
- Intestinal microbial variation may predict early acute rejection after liver transplantation in rats. [Journal Article]
- Transplantation 2014 Oct 27; 98(8):844-52.
Acute rejection (AR) remains a life-threatening complication after orthotopic liver transplantation (OLT) and there are few available diagnostic biomarkers clinically for AR. This study aims to identify intestinal microbial profile and explore potential application of microbial profile as a biomarker for AR after OLT.The OLT models in rats were established. Hepatic graft histology, ultrastructure, function, and intestinal barrier function were tested. Ileocecal contents were collected for intestinal microbial analysis.Hepatic graft suffered from the ischemia-reperfusion (I/R) injury on day 1, initial AR on day 3, and severe AR on day 7 after OLT. Real-time quantitative polymerase chain reaction results showed that genus Faecalibacterium prausnitzii and Lactobacillus were decreased, whereas Clostridium bolteae was increased during AR. Notably, cluster analysis of denaturing gradient gel electrophoresis (DGGE) profiles showed the 7AR and 3AR groups clustered together with 73.4% similarity, suggesting that intestinal microbiota was more sensitive than hepatic function in responding to AR. Microbial diversity and species richness were decreased during AR. Phylogenetic tree analysis showed that most of the decreased key bacteria belonged to phylum Firmicutes, whereas increased key bacteria belonged to phylum Bacteroidetes. Moreover, intestinal microvilli loss and tight junction damage were noted, and intestinal barrier dysfunction during AR presented a decrease of fecal secretory immunoglobulin A (sIgA) and increase of blood bacteremia, endotoxin, and tumor necrosis factor-α.We dynamically detail intestinal microbial characterization and find a high sensitivity of microbial change during AR after OLT, suggesting that intestinal microbial variation may predict AR in early phase and become an assistant therapeutic target to improve rejection after OLT.