Online Measurement System for Dynamic Flow Bioreactors to Study Barrier Integrity of hiPSC-Based Blood–Brain Barrier In Vitro Models

Electrochemical impedance spectroscopy (EIS) is a noninvasive, reliable, and efficient method to analyze the barrier integrity of in vitro tissue models. This well-established tool is used most widely to quantify the transendothelial/epithelial resistance (TEER) of Transwell-based models cultured under static conditions. However, dynamic culture in bioreactors can achieve advanced cell culture conditions that mimic a more tissue-specific environment and stimulation. This requires the development of culture systems that also allow for the assessment of barrier integrity under dynamic conditions. Here, we present a bioreactor system that is capable of the automated, continuous, and non-invasive online monitoring of cellular barrier integrity during dynamic culture. Polydimethylsiloxane (PDMS) casting and 3D printing were used for the fabrication of the bioreactors. Additionally, attachable electrodes based on titanium nitride (TiN)-coated steel tubes were developed to perform EIS measurements. In order to test the monitored bioreactor system, blood–brain barrier (BBB) in vitro models derived from human-induced pluripotent stem cells (hiPSC) were cultured for up to 7 days. We applied equivalent electrical circuit fitting to quantify the electrical parameters of the cell layer and observed that TEER gradually decreased over time from 2513 Ω·cm2 to 285 Ω·cm2, as also specified in the static control culture. Our versatile system offers the possibility to be used for various dynamic tissue cultures that require a non-invasive monitoring system for barrier integrity.

Gardnerella Vaginalis Alters Cervicovaginal Epithelial Cell Function Through Epithelial Cell-type Specific Immune Responses

Background: The cervicovaginal (CV) microbiome is highly associated with vaginal health and disease in both pregnant and non-pregnant individuals. An overabundance of Gardnerella vaginalis in the CV space is commonly associated with adverse reproductive outcomes including bacterial vaginosis (BV), sexually transmitted diseases and preterm birth while the presence of Lactobacillus spp is often associated with reproductive health. While host-microbial interactions are hypothesized to contribute to CV health and disease, the mechanisms by which these interactions regulate CV epithelial function remain largely unknown. Results: Using an in vitro co-culture model, we assessed the effects of Lactobacillus crispatus and G. vaginalis on the CV epithelial barrier, the immune mediators that could be contributing to decreased barrier integrity and the immune signaling pathways regulating the immune response. G. vaginalis, but not L. crispatus, significantly increased epithelial cell death and decreased epithelial barrier integrity in an epithelial cell-specific manner. A G. vaginalis-mediated epithelial immune response including NFkB activation and proinflammatory cytokine release was initiated partially through TLR2 dependent signaling pathways. Additionally, investigation of the cytokine immune profile in human CV fluid showed distinctive clustering of cytokines by G. vaginalis abundance and birth outcome. Conclusions: The results of this study show both microbe- and epithelial cell-type specific effects on CV epithelial function. Altered epithelial barrier function through cell death and immune mediated mechanisms by G. vaginalis, but not L. crispatus, indicates that host epithelial cells respond to bacteria-associated signals, resulting in altered epithelial function and ultimately CV disease. Additionally, distinct immune signatures associated with G. vaginalis or birth outcome provide further evidence that host-microbial interactions may contribute significantly to the biological mechanisms regulating reproductive outcomes.

The use of exogenous enzymes to optimize gut function in pigs

Exogenous enzymes are used in pig diets to improve the availability and digestibility of some non-accessible nutrients. As result of this enhanced digestion, short fragments of these molecules may become available in the distal foregut and the hindgut and modulate microbiota composition, gut barrier integrity, and overall animal health. This chapter reviews the effects of different exogenous enzymes (carbohydrases, phytases, proteases and lipases) on nutrient digestibility, gut microbial ecology, and barrier function and immunity of pigs at different ages (sows, weaned piglets, growing/fattening pigs). Exogenous enzymes are usually included into feeds as blends so they can complement each other’s activities and further improve the accessibility to non-digestible structures. Exogenous enzymes used in feed manufacturing for more than 30 years, initially to improve the digestive function of non-digestible nutrients (i.e. fibre, phytic acid, etc.), more recently other indirect actions on the regulation of gut microbiota and gut health have gained interest.

GPR120 prevents colorectal adenocarcinoma progression by sustaining the mucosal barrier integrity

GPR120 (encoded by FFAR4 gene) is a receptor for long chain fatty acids, activated by ω-3 Polyunsaturated Fatty Acids (PUFAs), and expressed in many cell types. Its role in the context of colorectal cancer (CRC) is still puzzling with many controversial evidences. Here, we explored the involvement of epithelial GPR120 in the CRC development. Both in vitro and in vivo experiments were conducted to mimic the conditional deletion of the receptor from gut epithelium. Intestinal permeability and integrity of mucus layer were assessed by using Evans blue dye and immunofluorescence for MUC-2 protein, respectively. Microbiota composition, presence of lipid mediators and short chain fatty acids were analyzed in the stools of conditional GPR120 and wild type (WT) mice. Incidence and grade of tumors were evaluated in all groups of mice before and after colitis-associated cancer. Finally, GPR120 expression was analyzed in 9 human normal tissues, 9 adenomas, and 17 primary adenocarcinomas. Our work for the first time highlights the role of the receptor in the progression of colorectal cancer. We observed that the loss of epithelial GPR120 in the gut results into increased intestinal permeability, microbiota translocation and dysbiosis, which turns into hyperproliferation of epithelial cells, likely through the activation of β -catenin signaling. Therefore, the loss of GPR120 represents an early event of CRC, but avoid its progression as invasive cancer. these results demonstrate that the epithelial GPR120 receptor is essential to maintain the mucosal barrier integrity and to prevent CRC developing. Therefore, our data pave the way to GPR120 as an useful marker for the phenotypic characterization of CRC lesions and as new potential target for CRC prevention.

YES kinase controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis

Vascular Endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in progression of diseases such as cancer and eye diseases. Inhibitors of SRC cytoplasmic tyrosine kinase have been applied to suppress tyrosine phosphorylation of VE-cadherin and thereby to prevent excessive leakage, edema and high interstitial pressure. We show that the SRC-related YES tyrosine kinase rather than SRC, is localized at endothelial cell (EC) junctions. EC-specific YES deletion suppresses VE-cadherin phosphorylation, and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration, and exaggerated agonist-induced macromolecular leakage, while extravasation of monocytes is suppressed. Overexpression of Yes causes ectopic VE-cadherin phosphorylation while vascular leakage is unaffected. In contrast, in EC-specific Src-deficient mice, VE-cadherin internalization is maintained and leakage is suppressed. In conclusion, YES-mediated VE-cadherin phosphorylation regulates its constitutive turnover, required for endothelial junction plasticity and vascular integrity.

Protective Effect of Probiotics Isolated from Traditional Fermented Tea Leaves (Miang) from Northern Thailand and Role of Synbiotics in Ameliorating Experimental Ulcerative Colitis in Mice

This study aimed to investigate the protective effect of probiotics and synbiotics from traditional Thai fermented tea leaves (Miang) on dextran sulfate sodium (DSS)-induced colitis in mice, in comparison to sulfasalazine. C57BL/6 mice were treated with probiotics L. pentosus A14-6, CMY46 and synbiotics, L. pentosus A14-6 combined with XOS, and L. pentosus CMY46 combined with GOS for 21 days. Colitis was induced with 2% DSS administration for seven days during the last seven days of the experimental period. The positive group was treated with sulfasalazine. At the end of the experiment, clinical symptoms, pathohistological changes, intestinal barrier integrity, and inflammatory markers were analyzed. The probiotics and synbiotics from Miang ameliorated DSS-induced colitis by protecting body weight loss, decreasing disease activity index, restoring the colon length, and reducing pathohistological damages. Furthermore, treatment with probiotics and synbiotics improved intestinal barrier integrity, accompanied by lowing colonic and systemic inflammation. In addition, synbiotics CMY46 combined with GOS remarkedly elevated the expression of IL-10. These results suggested that synbiotics isolated from Miang had more effectiveness than sulfasalazine. Thereby, they could represent a novel potential natural agent against colonic inflammation.

Hypoxia increases expression of selected blood–brain barrier transporters GLUT-1, P-gp, SLC7A5 and TFRC, while maintaining barrier integrity, in brain capillary endothelial monolayers

Background Brain capillary endothelial cells (BCECs) experience hypoxic conditions during early brain development. The newly formed capillaries are tight and functional before astrocytes and pericytes join the capillaries and establish the neurovascular unit. Brain endothelial cell phenotype markers P-gp (ABCB1), LAT-1(SLC7A5), GLUT-1(SLC2A1), and TFR(TFRC) have all been described to be hypoxia sensitive. Therefore, we hypothesized that monolayers of BCECs, cultured under hypoxic conditions, would show an increase in LAT-1, GLUT-1 and TFR expression and display tight endothelial barriers. Methods and results Primary bovine BCECs were cultured under normoxic and hypoxic conditions. Chronic hypoxia induced HIF-1α stabilization and translocation to the nucleus, as judged by immunocytochemistry and confocal laser scanning imaging. Endothelial cell morphology, claudin-5 and ZO-1 localization and barrier integrity were unaffected by hypoxia, indicating that the tight junctions in the BBB model were not compromised. SLC7A5, SLC2A1, and TFRC-mRNA levels were increased in hypoxic cultures, while ABCB1 remained unchanged as shown by real-time qPCR. P-gp, TfR and GLUT-1 were found to be significantly increased at protein levels. An increase in uptake of [3H]-glucose was demonstrated, while a non-significant increase in the efflux ratio of the P-gp substrate [3H]-digoxin was observed in hypoxic cells. No changes were observed in functional LAT-1 as judged by uptake studies of [3H]-leucine. Stabilization of HIF-1α under normoxic conditions with desferrioxamine (DFO) mimicked the effects of hypoxia on endothelial cells. Furthermore, low concentrations of DFO caused an increase in transendothelial electrical resistance (TEER), suggesting that a slight activation of the HIF-1α system may actually increase brain endothelial monolayer tightness. Moreover, exposure of confluent monolayers to hypoxia resulted in markedly increase in TEER after 24 and 48 h, which corresponded to a higher transcript level of CLDN5. Conclusions Our findings collectively suggest that hypoxic conditions increase some BBB transporters' expression via HIF-1α stabilization, without compromising monolayer integrity. This may in part explain why brain capillaries show early maturation, in terms of barrier tightness and protein expression, during embryogenesis, and provides a novel methodological tool for optimal brain endothelial culture.