Monolayer Permeability Research Articles

Bifidobacterium bifidum Strain BB1 Inhibits Tumor Necrosis Factor-α–Induced Increase in Intestinal Epithelial Tight Junction Permeability via Toll-Like Receptor-2/Toll-Like Receptor-6 Receptor Complex–Dependent Stimulation of Peroxisome Proliferator-Activated Receptor γ and Suppression of NF-κB p65

Bifidobacterium bifidum (BB) strain BB1 causes a strain-specific enhancement in intestinal epithelial tight junction (TJ) barrier. TNF-α induces an increase in intestinal epithelial TJ permeability and promotes intestinal inflammation. The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to unravel the intracellular mechanisms involved. Previously reported, TNF-α produces an increase in intestinal epithelial TJ permeability in Caco-2 monolayers and in mice. The addition of BB1 inhibited the TNF-α increase in Caco-2 intestinal TJ permeability and mouse intestinal permeability in a strain-specific manner. BB1 inhibited the TNF-α induced increase in intestinal TJ permeability by interfering the with TNF-α induced enterocyte NF-κB p50/p65 and MLCK gene activation. The BB1 protective effect against the TNF-α induced increase in intestinal permeability was mediated by TLR-2/TLR-6 heterodimer complex activation of PPAR-γ and PPAR-γ pathway inhibition of TNF-α induced IKK-α activation, which in turn resulted in a step-wise inhibition of NF-κB p50/p65, MLCK gene, MLCK kinase activity, MLCK-induced opening of the TJ barrier. In conclusion, these studies unravel novel intracellular mechanisms of BB1 protection against the TNF-α induced increase in intestinal TJ permeability. Our data show that BB1 protects against the TNF-α induced increase in intestinal epithelial TJ permeability via a PPAR-γ dependent inhibition of NF-κB p50/p65 and MLCK gene activation.

Prevotella melaninogenica disrupted oral epithelial barrier function via myosin light chain kinase.

Our previous studies have found that the composition ratio of Prevotella melaninogenica (Pm) on buccal mucosa surface of oral lichen planus (OLP) patients increased significantly compared with control. Furthermore, Pm could invade the epithelium of OLP patients. This study aimed to further explore the impact of Pm on oral keratinocytes. The Pm-human oral keratinocyte (HOK) co-culture model was established to detect monolayer permeability, zona occludens-1 (ZO-1) expression, and intracellular survival of Pm. We performed RNA-seq followed by identification of differentially expressed genes (DEGs) and Gene Ontology (GO) analysis, with a particular focus on myosin light chain kinase (MLCK). An MLCK inhibitor ML-7 was utilized in Pm-HOK co-culture model to assess its effects on monolayer permeability and ZO-1 expression. HOK monolayer permeability was increased, and ZO-1 expression was decreased after co-culture (p < 0.05). Pm could survive in HOK cells. RNA-seq revealed MLCK was an upregulated common DEG. The expression of MLCK in the Pm-HOK co-culture model was upregulated. Inhibition of MLCK rescued the increased epithelial permeability, and ZO-1 expression was upregulated (p < 0.05). MLCK may be involved in disrupting epithelial barrier function by Pm.

Semaphorin 7A promotes endothelial permeability and inflammation via plexin C1 and integrin β1 in Kawasaki disease

BackgroundKawasaki disease (KD) is a pediatric systemic vasculitis characterized by endothelial cell dysfunction. Semaphorin 7A (Sema7A) has been reported to regulate endothelial phenotypes associated with cardiovascular diseases, while its role in KD remains unknown. This study aims to investigate the effect of Sema7A on endothelial permeability and inflammatory response in KD conditions.MethodsBlood samples were collected from 68 KD patients and 25 healthy children (HC). The levels of Sema7A and A Disintegrin and Metalloprotease 17 (ADAM17) in serum were measured by enzyme-linked immunosorbent assay (ELISA), and Sema7A expression in blood cells was analyzed by flow cytometry. Ex vivo monocytes were used for Sema7A shedding assays. In vitro human coronary artery endothelial cells (HCAECs) were cultured in KD sera and stimulated with Sema7A, and TNF-α, IL-1β, IL-6, and IL-18 of HCAECs were measured by ELISA and qRT-PCR. HCAECs monolayer permeability was measured by FITC-dextran.ResultsThe serum level of Sema7A was significantly higher in KD patients than in HC and correlated with disease severity. Monocytes were identified as one of the source of elevated serum Sema7A, which implicates a process of ADAM17-dependent shedding. Sera from KD patients induced upregulation of plexin C1 and integrin β1 in HCAECs compared to sera from HC. Sema7A mediated the proinflammatory cytokine production of HCAECs in an integrin β1-dependent manner, while both plexin C1 and integrin β1 contributed to Sema7A-induced HCAEC hyperpermeability.ConclusionsSema7A is involved in the progression of KD vasculitis by promoting endothelial permeability and inflammation through a plexin C1 and integrin β1-dependent pathway. Sema7A may serve as a potential biomarker and therapeutic target in the prognosis and treatment of KD.

ATP13A3 variants promote pulmonary arterial hypertension by disrupting polyamine transport.

Potential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in patients with pulmonary arterial hypertension (PAH). ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. In this study, we sought to determine the biological function of ATP13A3 in vascular endothelial cells (ECs) and how PAH-associated variants may contribute to disease pathogenesis. We studied the impact of ATP13A3 deficiency and overexpression in EC models [human pulmonary ECs, blood outgrowth ECs (BOECs), and human microvascular EC 1], including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. ATP13A3 localized to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation, and increased monolayer permeability to thrombin. The assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, and L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germline Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling, and muscularization of pulmonary vessels. We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, a deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH.

428 Promoting Infant Gut Barrier Development Through Culturally Relevant Adoption of Fruit and Vegetable Intake.

OBJECTIVES/GOALS: To determine in vitro mechanisms by which fruits and vegetables (FV) contribute to colon barrier development in Latin American infants. We hypothesize that simulated colonic fermentation of FVs will stimulatein vitro cell barrier function by activating the hypoxia-inducible factor (HIF) pathway in colonocytes. METHODS/STUDY POPULATION: FVs consumed by US-based Latin American infants 6-12 months old (identified from NHANES-What We Eat in America Surveys) will be combined with human breast-milk samples from women self-identified as Hispanic or non-Hispanic, and then subjected to in vitro digestion and anaerobic colonic fermentation using human feces. FV fermenta will be incubated with Caco2 monolayers to measure in vitro cell permeability and protein levels of cellular tight junction, metabolic, and HIF signaling enzymes. To examine their effects in vivo, FVs identified to modulate in vitro barrier function, will be fed (5% freeze dried powder) to wild-type mice and the above parameters will be examined. If in vivo effects are found, intestinal specific HIF knockout mice will be used to examine the role of HIF signaling in mediating these effects. RESULTS/ANTICIPATED RESULTS: We expect that fermenta derived from human milk and FVs will reduce in vitro gut permeability in Caco2 monolayers by increasing gene and protein expression of the HIF signaling complex relative to fermenta of human milk alone. This will be reflected with higher cellular trans-epithelial resistance and greater expression levels of tight junction proteins. We expect FV powder consumption will similarly increase in vivo gut permeability and expression of related genes in mice as compared to mice fed diets without FVs. As we expect an increase in HIF signaling in the colon, we expect that FV powder consumption will not enhance in vivo gut permeability in mice colons with an intestinal specific knockout of HIF. DISCUSSION/SIGNIFICANCE: Data from this study will provide mechanistic evidence to help clinicians promote relevant FVs recommendations for Latin American infants and families. Due to the link between gut permeability and obesity, our next step will be to conduct a dietary intervention in this population.

Lifibrate attenuates blood-brain barrier damage following ischemic stroke via the MLCK/p-MLC/ZO-1 axis.

Dysfunction of tight junction proteins-associated damage to the blood-brain barrier (BBB) plays an important role in the pathogenesis of ischemic stroke. Lifibrate, an inhibitor of cholinephosphotransferase (CPT), has been used as an agent for serum lipid lowering. However, the protective effects of Lifibrate in ischemic stroke and the underlying mechanism have not been clearly elucidated. Here, we employed an in vivo mice model of MCAO and an OGD/R model in vitro. In the mice models, neurological deficit scores and infarct volume were assessed. Evans Blue solution was used to detect the BBB permeability. The TEER was examined to determine brain endothelial monolayer permeability. Here, we found that Lifibrate improved neurological dysfunction in stroke. Additionally, increased BBB permeability during stroke was significantly ameliorated by Lifibrate. Correspondingly, the reduced expression of the tight junction protein ZO-1 was restored by Lifibrate at both the mRNA and protein levels. Using an in vitro model, we found that Lifibrate ameliorated OGD/R-induced injury in human bEnd.3 brain microvascular endothelial cells by increasing cell viability but reducing the release of LDH. Importantly, Lifibrate suppressed the increase in endothelial monolayer permeability and the reduction in TEER induced by OGD/R via the rescue of ZO-1 expression. Mechanistically, Lifibrate blocked activation of the MLCK/ p-MLC signaling pathway in OGD/R-stimulated bEnd.3 cells. In contrast, overexpression of MLCK abolished the protective effects of Lifibrate in endothelial monolayer permeability, TEER, as well as the expression of ZO-1. Our results provide a basis for further investigation into the neuroprotective mechanism of Lifibrate during stroke.

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina.

Diabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, leading to visual impairment. Retinal pigment epithelium (RPE) injury is a key component of the outer blood retinal barrier, and its damage is an important indicator of DR. Receptor for activated C kinase 1 (RACK1) activates protein kinase C-ε (PKC-ε) to promote the generation of reactive oxygen species (ROS) in RPE cells, leading to apoptosis. Therefore, we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS, thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR. To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR. In this study, Sprague-Dawley rats and adult RPE cell line-19 (ARPE-19) cells were used as in vivo and in vitro models, respectively, to explore the role of RACK1 in mediating PKC-ε in early DR. Furthermore, the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model. Streptozotocin-induced diabetic rats showed increased apoptosis and up-regulated expression of RACK1 and PKC-ε proteins in RPE cells following a prolonged modeling. Similarly, ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε, accompanied by an increases in ROS production, apoptosis rate, and monolayer permeability. However, silencing RACK1 significantly downregulated the expression of PKC-ε and ROS, reduced cell apoptosis and permeability, and protected barrier function. RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Functional roles of CD26/DPP4 in lipopolysaccharide-induced lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by dysregulated inflammation and increased permeability of lung microvascular cells. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein that is expressed in several cell types and mediates multiple pleiotropic effects. We previously reported that DPP4 inhibition by sitagliptin attenuates lipopolysaccharide (LPS)-induced lung injury in mice. The current study characterized the functional role of CD26/DPP4 expression in LPS-induced lung injury in mice, isolated alveolar macrophages, and cultured lung endothelial cells. In LPS-induced lung injury, inflammatory responses [bronchoalveolar lavage fluid (BALF) neutrophil numbers and several proinflammatory cytokine levels] were attenuated in Dpp4 knockout (Dpp4 KO) mice. However, multiple assays of alveolar capillary permeability were similar between the Dpp4 KO and wild-type mice. TNF-α and IL-6 production was suppressed in alveolar macrophages isolated from Dpp4 KO mice. In contrast, in cultured mouse lung microvascular endothelial cells (MLMVECs), reduction in CD26/DPP4 expression by siRNA resulted in greater ICAM-1 and IL-6 expression after LPS stimulation. Moreover, the LPS-induced vascular monolayer permeability in vitro was higher in MLMVECs treated with Dpp4 siRNA, suggesting that CD26/DPP4 plays a protective role in endothelial barrier function. In summary, this study demonstrated that genetic deficiency of Dpp4 attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential functional roles of CD26/DPP4 expression in resident cellular components of the lung. CD26/DPP4 may be a potential therapeutic target for ARDS and warrants further exploration to precisely identify the multiple functional effects of CD26/DPP4 in ARDS pathophysiology.NEW & NOTEWORTHY We aimed to clarify the functional roles of CD26/DPP4 in ARDS pathophysiology using Dpp4-deficient mice and siRNA reduction techniques in cultured lung cells. Our results suggest that CD26/DPP4 expression plays a proinflammatory role in alveolar macrophages while also playing a protective role in the endothelial barrier. Dpp4 genetic deficiency attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential roles of CD26/DPP4 expression in the resident cellular components of the lung.

Azilsartan improves urinary albumin excretion in hypertension mice.

Hypertension is one of the most important risk factors for chronic kidney diseases, leading to hypertensive nephrosclerosis, including excessive albuminuria. Azilsartan, an angiotensin II type 1 receptor blocker, has been widely used for the treatment of hypertension. However, the effects of Azilsartan on urinary albumin excretion in hypertension haven't been reported before. In this study, we investigated whether Azilsartan possesses a beneficial property against albuminuria in mice treated with angiotensin II and a high-salt diet (ANG/HS). Compared to the control group, the ANG/HS group had higher blood pressure, oxidative stress, and inflammatory response, all of which were rescued by Azilsartan dose-dependently. Importantly, the ANG/HS-induced increase in urinary albumin excretion and decrease in the expression of occludin were reversed by Azilsartan. Additionally, it was shown that increased fluorescence intensity of FITC-dextran, declined trans-endothelial electrical resistance (TEER) values, and reduction of occludin and krüppel-like factor 2 (KLF2) were observed in ANG/HS-treated human renal glomerular endothelial cells (HrGECs), then prevented by Azilsartan. Moreover, the regulatory effect of Azilsartan on endothelial monolayer permeability in ANG/HS-treated HrGECs was abolished by the knockdown of KLF2, indicating KLF2 is required for the effect of Azilsartan. We concluded that Azilsartan alleviated diabetic nephropathy-induced increase in Uterine artery embolization (UAE) mediated by the KLF2/occludin axis.

Dietary pyrroloquinoline quinone hinders aging progression in male mice and D-galactose-induced cells.

Background: Understanding and promoting healthy aging has become a necessity in the modern world, where life expectancy is rising. The prospective benefits of the antioxidant pyrroloquinoline quinone (PQQ) in healthy aging are promising. However, its role in aging remains unclear. Thus, this study aimed to investigate the effect of PQQ on preventing the progression of aging and to explore its underlying molecular mechanisms. Methods: Naturally aged C57BL/6J male mice were fed a normal diet with or without PQQ (20mg/kg/day) for 10 weeks. Body composition was measured by bioimpedance at weeks 0 and 8. The integument conditions were evaluated at weeks 0, 4, and 8. Muscle strength and function were examined at week 8. At the ninth week, computed tomography images of the mice were captured, and blood and tissue samples were collected. The levels of inflammatory cytokines in the gastrocnemius muscle were measured, and the muscle fiber cross-sectional area in the soleus muscle was examined. Additionally, a D-galactose (D-gal)-induced cell aging model was used to study the effects of PQQ intervention on cell proliferation, senescence, differentiation, ROS levels, and mitochondrial function in myoblasts (C2C12). Cell proliferation and monolayer permeability of D-gal-induced intestinal epithelial cells (IEC6) were also examined. Results: Aged mice suffered from malnutrition; however, PQQ supplementation ameliorated this effect, possibly by improving metabolic dysfunction and small intestinal performance. PQQ prevented rapid loss of body fat and body fluid accumulation, attenuated muscle atrophy and weakening, reduced chronic inflammation in skeletal muscles, and improved skin and coating conditions in aged mice. Furthermore, PQQ intervention in D-gal-treated C2C12 cells improved mitochondrial function, reduced cellular reactive oxygen species (ROS) levels and senescence, and enhanced cell differentiation, consequently preventing age-related muscle atrophy. In addition, PQQ increased cell proliferation in D-gal-treated IEC6 cells and consequently improved intestinal barrier function. Conclusion: PQQ could hinder the aging process and particularly attenuate muscle atrophy, and muscle weakness by improving mitochondrial function, leading to reduced age-related oxidative stress and inflammation in muscles. PQQ may also ameliorate malnutrition caused by intestinal barrier dysfunction by enhancing IEC proliferation. This study provides evidence for the role of PQQ in aging and suggests that PQQ may be a potential nutritional supplementation that can be included in healthy aging strategies.

Tritordeum: Promising Cultivars to Improve Health.

Tritordeum is an amphiploides species resulting from the hybridization between durum wheat (T. durum) and wild barley (H. chilense). This new cereal is considered a natural crop as it is obtained by traditional breeding techniques. Given its appreciable organoleptic characteristics, agronomic features, presence of interesting components, and good technological properties, Tritordeum is of promising interest for the development of health-oriented foods. In this study, we evaluated two registered Tritordeum cultivars, Bulel and Aucan. T. durum (Provenzal) was employed as the positive control. The extracted proteins were digested by gastric/pancreatic proteases, and their biological effects on Caco-2 differentiated on transwell inserts were determined. Changes in cell viability, monolayer permeability, organization of F-actin microfilaments, and ER stress triggered by protein-digested samples (DPs) were inspected. Our results showed that exposure to Provenzal-DPs promptly disrupted the tight junction barrier. Conversely, Aucan-DPs did not enhance monolayer permeability, whereas Bulel-DPs exerted only slight effects. Provental-DPs-induced toxicity was also confirmed by changes in cell viability and by the deep reorganization of the enterocyte cytoskeleton. In contrast, Aucan-DPs and Bulel-DPs did not affect monolayer viability and cytoskeleton structure. Overall, our findings suggest that both Tritordeum cultivars could be potential candidates for mitigating the toxicity of wheat flour.

Genetic and Phenotypic Characterization of Bacillus velezensis Strain BV379 for Human Probiotic Applications.

Bacterial spore-forming Bacillaceae species, including Bacillus subtilis and Heyndrickxia coagulans, are increasingly utilized for probiotic dietary supplementation. Bacillus velezensis is a Bacillus species that is frequently used as a direct-fed microbial in animal feed but less so as a probiotic for humans. The objective of this study was to characterize the suitability of the Bacillus velezensis strain BV379 for probiotic applications by (1) in silico screening for both adverse genetic elements and putatively beneficial traits, (2) in vitro evaluation of interactions with human intestinal epithelial cells, and (3) in vitro characterization of BV379 spore viability at various temperatures, pH, and in the presence of bile salt. In silico screening of the BV379 genome revealed few genes encoding Bacillaceae-associated toxins, virulence factors, and enzymes involved in the production of toxins. While BV379 encodes five antimicrobial resistance genes, minimum inhibitory concentration assays determined that BV379 is susceptible to all eight clinically relevant antibiotics tested. Preliminary cell culture experiments showed that BV379 lysates did not adversely impact human intestinal epithelial cell viability and monolayer permeability. It was also determined that BV379 spores can easily tolerate the harsh pH, bile salt, and microaerobic conditions typical of the GI tract. Altogether, the results presented herein support the safety and potential of Bacillus velezensis strain BV379 for use as an oral probiotic.

Effect of Latanoprostene Bunod on the Permeability of Trabecular Meshwork Cells

Purpose: To investigate the effects of latanoprostene bunod (LBN) on nitric oxide (NO) production and permeability in human trabecular meshwork cells (HTMC).Methods: HTMC were treated with 50 and 100 µM LBN and latanoprost free acid (LAT) for 30 minutes. Additionally, 100 µM LBN was co-exposed to 0.5 mM L-NAME (N-Nitroarginine methyl ester). Cellular viability and NO production were measured using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) and Griess assays. The permeability and resistance of the HTMC monolayer were evaluated by trans-endothelial electrical resistance (TEER) and carboxyfluorescein permeability.Results: Exposure to 100 µM LBN led to increased NO production, whereas co-exposure to L-NAME reduced NO production. Treatment with 100 µM LBN decreased the TEER of the HTMC monolayer. LBN exposure heightened carboxyfluorescein permeability, but co-exposure to 100 µM LBN and L-NAME reduced permeability. LAT treatment did not affect NO production or permeability.Conclusions: LBN increased the permeability of the HTMC monolayer and increased NO production. Therefore, LBN might increase trabecular outflow in addition to promoting uveoscleral outflow.

P111 Dietary emulsifier κ-carrageenan does not directly affect intestinal permeability in organoid-derived epithelial monolayers from patients with Crohn’s disease

Abstract Background The dietary emulsifier carrageenan (CGN) is widely used to optimise the texture of processed foods such as ice cream, plant-based milk and processed meat products. However, CGN is suggested to play a role in IBD development by exerting pro-inflammatory effects and increasing intestinal permeability, as shown in immortalized cell lines and murine models. Specifically kappa-carrageenan (κ-CGN) has been shown to disrupt barrier function of intestinal epithelial cells. In this study, we investigated if κ-CGN can directly increase intestinal permeability in organoid-derived epithelial monolayers from patients with Crohn’s disease (CD). Methods Organoid cultures were derived from colonic biopsies of 5 patients with CD (Table 1) and seeded on Transwell® inserts to obtain epithelial monolayers. Two independent experiments were performed per patient (n = 10). Once a confluent monolayer was formed, non-inflamed and inflamed conditions were established by adding regular culture medium or inflammatory stimuli (100 ng/mL TNF-α, 20 ng/mL IL-1β, 1 µg/mL flagellin) to the basolateral side, respectively. The next day, both inflamed and non-inflamed monolayers were stimulated with 100 µg/mL κ-CGN on the apical side, or with regular culture medium as control. The basolateral side was also renewed with either the inflammatory stimuli or regular culture medium. Transepithelial electrical resistance (TEER) was measured after 24 and 48 hours of κ-CGN exposure to study permeability of the epithelium. Relative TEER (% change compared to baseline) was compared between controls and κ-CGN stimulated conditions (repeated measures ANOVA, Šídák's multiple comparisons correction). Results As expected, exposure to inflammatory stimuli on the basolateral side resulted in a significant reduction of TEER of the monolayers after 48 hours (+ 24 hours pre-stimulation), compared to the non-inflamed conditions (-22.54 ± 4.557 %, p&amp;lt;0.0001, paired t-test) (Figure 1A). After 24 hours of stimulation with κ-CGN, TEER did not significantly change in both the non-inflamed (p=0.90) and inflamed conditions (p=0.80), compared to the control conditions. Also after 48 hours, exposure to κ-CGN did not significantly affect the TEER in both non-inflamed (p=0.94) and inflamed (p=0.74) conditions (Figure 1B). RNA analysis of the organoid-derived monolayers is ongoing to explore the inflammatory potential of κ-CGN and possible effects on mucus production. Conclusion The dietary emulsifier κ-CGN does not directly alter the permeability of the intestinal epithelium of CD patients. Further analysis of gene expression of inflammatory markers and mucus production is ongoing. Possible indirect effects, through alterations of the microbiota or immune cells, should still be explored.

Novel Bacteroides Vulgatus strain protects against gluten-induced break of human celiac gut epithelial homeostasis: a pre-clinical proof-of-concept study

Background and aimsWe have identified a decreased abundance of microbial species known to have a potential anti-inflammatory, protective effect in subjects that developed Celiac Disease (CeD) compared to those who did not. We aim to confirm the potential protective role of one of these species, namely Bacteroides vulgatus, and to mechanistically establish the effect of bacterial bioproducts on gluten-dependent changes on human gut epithelial functions.MethodsWe identified, isolated, cultivated, and sequenced a unique novel strain (20220303-A2) of B. vulgatus found only in control subjects. Using a human gut organoid system developed from pre-celiac patients, we monitored epithelial phenotype and innate immune cytokines at baseline, after exposure to gliadin, or gliadin plus B. vulgatus cell free supernatant (CFS).ResultsFollowing gliadin exposure, we observed increases in epithelial cell death, epithelial monolayer permeability, and secretion of pro-inflammatory cytokines. These effects were mitigated upon exposure to B. vulgatus 20220303-A2 CFS, which had matched phenotype gene product mutations. These protective effects were mediated by epigenetic reprogramming of the organoids treated with B. vulgatus CFS.ConclusionsWe identified a unique strain of B. vulgatus that may exert a beneficial role by protecting CeD epithelium against a gluten-induced break of epithelial tolerance through miRNA reprogramming.ImpactGut dysbiosis precedes the onset of celiac disease in genetically at-risk infants.This dysbiosis is characterized by the loss of protective bacterial strains in those children who will go on to develop celiac disease.The paper reports the mechanism by which one of these protective strains, B. vulgatus, ameliorates the gluten-induced break of gut epithelial homeostasis by epigenetically re-programming the target intestinal epithelium involving pathways controlling permeability, immune response, and cell turnover.

A novel trypsin of Trichinella spiralis mediates larval invasion of gut epithelium via binding to PAR2 and activating ERK1/2 pathway.

Proteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved. TsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo. TsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

Unraveling the Action Mechanism of Tubeimoside-1 against Tumor Microvessels via Network Pharmacology and Experimental Validation.

Objective: Tubeimoside-1 (TBMS1) is a plant-derived triterpenoid saponin that exhibits pharmacological properties and anti-tumor effects, but the anti-tumor microvessels of action of TBMS1 remains to be completely elucidated. This study aims to verify the effect of TBMS1 on tumor microvessels and its underlying mechanism. Methods: A SKOV3 xenografted mouse model were constructed to evaluate the anti-tumor microvessels of TBMS1 in vivo, followed by function assays to verify the effects of TBMS1 on the proliferation, cell cycle, migration, and tubule formation of vascular endothelial cells in vitro. Next, based on network pharmacology, the drug/disease-target protein-protein interaction (PPI) networks, biological functions and gene enrichment analyses were performed to predict the underlying mechanism. Finally, molecules and pathways associated with tumor trans-endothelial migration were identified. Results: TBMS1 treatment effectively reduced tumor microvessel density in ovarian cancer model and inhibited the proliferation, cell cycle, migration, and induced apoptosis of vascular endothelial cells in vitro. Network pharmacological data suggested that tumor cell adhesion and trans-endothelial migration may participate in antiangiogenic effects of TBMS1. By endothelial adhesion and permeability assay, we identified that tumor adhesion and the permeability of endothelial monolayers were reduced by TBMS1. Furthermore, adhesion protein (VCAM-1and ICAM-1) and tight junction (TJ) proteins (VE-cadhsion, ZO-1 and claudin-5) were found to be regulated. Finally, Akt, Erk1/2, Stat3 and NF-κB signaling were decreased by TBMS1 treatment. Conclusion: To sum up, our findings strongly suggest that clinical application of TBSM1 may serve as a vasoactive drug treatment to suppress tumor progression.

A mechanical modeling framework to study endothelial permeability

The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighboring cells to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as thrombin, along with mechanical properties of the cell and extracellular matrix are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modeling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behavior that VE-cadherin complexes are known to exhibit, we model the VE-cadherin homophilic bond as cohesive contact with damage following a traction-separation law. We explicitly model the actin cytoskeleton and substrate to study their role in permeability. Our studies show that mechanochemical coupling is necessary to simulate the influence of the mechanical properties of the substrate on permeability. Simulations show that shear between cells is responsible for the variation in permeability between bicellular and tricellular junctions, explaining the phenotypic differences observed in experiments. An increase in the magnitude of traction force due to disturbed flow that endothelial cells experience results in increased permeability, and it is found that the effect is higher on stiffer extracellular matrix. Finally, we show that the cylindrical monolayer exhibits higher permeability than the planar monolayer under unconstrained cases. Thus, we present a contact mechanics-based mechanochemical model to investigate the variation in the permeability of endothelial monolayer due to multiple loads acting simultaneously.

Horizontal and vertical microchamber platforms for evaluation of the paracellular permeability of an epithelial cell monolayer.

Epithelial cells serve as a barrier by tightly adhering to each other and contribute to the homeostasis of living organisms by controlling substance permeation. Therefore, evaluation of the barrier function is important in pharmaceutical development processes. However, the widely used Transwell-based assays require the development of the defect-free epithelial cell monolayer above several tens of mm2, often resulting in low reproducibility and requiring a long incubation time. In addition, the culture surface of cells is far from the bottom of the well plate, making it difficult to observe the cell morphology using an optical microscope. Herein, we propose simple polydimethylsiloxane microfluidic devices for evaluating the barrier function of an epithelial monolayer using a microchamber array. After the formation of the epithelial monolayer over microchambers, the permeation of the marker molecules introduced above resulted in increased fluorescence intensity in microchambers, which was monitored using confocal laser scanning microscopy. We show that using this technique, alteration of the paracellular permeability induced by sodium caprate (C10) and cytochalasin-D, permeation enhancing factors, can be elucidated. Furthermore, by tilting the microchamber device 90 degrees, the vertical cell section and microchambers were imaged in the same focal plane, allowing for live visualization of the passage of fluorescent substances across the cell monolayer. This technique is expected to be useful for investigating the relationship between paracellular permeability and cell morphology, which is unattainable through conventional methods.