Fluorescein Isothiocyanate-dextran Research Articles

Ziziphus jujuba Miller Ethanol Extract Restores Disrupted Intestinal Barrier Function via Tight Junction Recovery and Reduces Inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition caused by the disruption of the intestinal barrier. The intestinal barrier is maintained by tight junctions (TJs), which sustain intestinal homeostasis and prevent pathogens from entering the microbiome and mucosal tissues. Ziziphus jujuba Miller (Z. jujuba) is a natural substance that has been used in traditional medicine as a therapy for a variety of diseases. However, in IBD, the efficacy of Z. jujuba is unknown. Therefore, we evaluated ZJB in Caco2 cells and a dextran sodium sulfate (DSS)-induced mouse model to demonstrate its efficacy in IBD. Z. jujuba extracts were prepared using 70% ethanol and were named ZJB. ZJB was found to be non-cytotoxic and to have excellent antioxidant effects. We confirmed its anti-inflammatory properties via the down-regulation of inflammatory factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). To evaluate the effects of ZJB on intestinal barrier function and TJ improvement, the trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran 4 kDa (FITC-Dextran 4) permeability were assessed. The TEER value increased by 61.389% and permeability decreased by 27.348% in the 200 μg/mL ZJB group compared with the 50 ng/mL IL-6 group after 24 h. Additionally, ZJB alleviated body weight loss, reduced the disease activity index (DAI) score, and induced colon shortening in 5% DSS-induced mice; inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were down-regulated in the serum. TJ proteins, such as Zonula occludens (ZO)-1 and occludin, were up-regulated by ZJB in an impaired Caco2 mouse model. Additionally, according to the liquid chromatography results, in tandem with mass spectrometry (LC-MS/MS) analysis, seven active ingredients were detected in ZJB. In conclusion, ZJB down-regulated inflammatory factors, protected intestinal barrier function, and increased TJ proteins. It is thus a safe, natural substance with the potential to be used as a therapeutic agent in IBD treatment.

High-fat diet induces sarcopenic obesity in natural aging rats through the gut–trimethylamine N-oxide–muscle axis

IntroductionThe lack of suitable animal models for sarcopenic obesity (SO) limits in-depth research into the disease. Emerging studies have demonstrated that gut dysbiosis is involved in the development of SO. As the importance of microbial metabolites is starting to unveil, it is necessary to comprehend the specific metabolites associated with gut microbiota and SO. ObjectivesWe aimed to investigate whether high-fat diet (HFD) causes SO in natural aging animal models and specific microbial metabolites that are involved in linking HFD and SO. MethodsYoung rats received HFD or control diet for 80 weeks, and obesity-related metabolic disorders and sarcopenia were measured. 16S rRNA sequencing and non-targeted and targeted metabolomics methods were used to detect fecal gut microbiota and serum metabolites. Gut barrier function was evaluated by intestinal barrier integrity and intestinal permeability. Trimethylamine N-oxide (TMAO) treatment was further conducted for verification. ResultsHFD resulted in body weight gain, dyslipidemia, impaired glucose tolerance, insulin resistance, and systemic inflammation in natural aging rats. HFD also caused decreases in muscle mass, strength, function, and fiber cross-sectional area and increase in muscle fatty infiltration in natural aging rats. 16S rRNA sequencing and nontargeted and targeted metabolomics analysis indicated that HFD contributed to gut dysbiosis, mainly characterized by increases in deleterious bacteria and TMAO. HFD destroyed intestinal barrier integrity and increased intestinal permeability, as evaluated by reducing levels of colonic mucin-2, tight junction proteins, goblet cells and elevating serum level of fluorescein isothiocyanate-dextran 4. Correlation analysis showed a positive association between TMAO and SO. In addition, TMAO treatment aggravated the development of SO in HFD-fed aged rats through regulating the ROS-AKT/mTOR signaling pathway. ConclusionHFD leads to SO in natural aging rats, partially through the gut–microbiota–TMAO–muscle axis.

Ketogenic Diet Protects from Experimental Colitis in a Mouse Model Regardless of Dietary Fat Source.

While ketogenic diets (KDs) may have potential as adjunct treatments for gastrointestinal diseases, there is little knowledge on how the fat source of these diets impacts intestinal health. The objective of this study was to investigate how the source of dietary fat of KD influences experimental colitis. We fed nine-week-old male C57BL/6J mice (n = 36) with a low-fat control diet or KD high either in saturated fatty acids (SFA-KD) or polyunsaturated linoleic acid (LA-KD) for four weeks and then induced colitis with dextran sodium sulfate (DSS). To compare the diets, we analyzed macroscopic and histological changes in the colon, intestinal permeability to fluorescein isothiocyanate-dextran (FITC-dextran), and the colonic expression of tight junction proteins and inflammatory markers. While the effects were more pronounced with LA-KD, both KDs markedly alleviated DSS-induced histological lesions. LA-KD prevented inflammation-related weight loss and the shortening of the colon, as well as preserved Il1b and Tnf expression at a healthy level. Despite no significant between-group differences in permeability to FITC-dextran, LA-KD mitigated changes in tight junction protein expression. Thus, KDs may have preventive potential against intestinal inflammation, with the level of the effect being dependent on the dietary fat source.

MiR-199a-5p modulates choroidal neovascularization by regulating Wnt7b/Wnt/β-catenin signaling pathway.

Choroidal neovascularization (CNV) can be seen in many fundus diseases, and lead to fundus exudation, bleeding, or vision loss. miRNAs are vital regulator in CNV. miR-199a-5p has been proved to be involved in regulating vascular formation of endothelial cells, but its role in CNV remains unclear. This study aims to study the role of miR-199a-5p in CNV. Laser irradiation was used to induce CNV model. The lesion area of CNV was calculated by high-resolution angiography with fluorescein isothiocyanate-dextran. Wnt family member 7b (Wnt7b), β-catenin, and Wnt pathway proteins was measured by western blot. Immunofluorescence was performed to test Wnt7b, β-catenin, CD31, and p-p65. miR-199a-5p and Wnt7b mRNA were tested by reverse transcription real-time polymerase chain reaction. Cell count kit-8, wound healing, Transwell, tube formation, and flow cytometry were used to detect the function of miR-199a-5p and Wnt7b on human retinal microvascular endothelial cells (HRMEC). TargetScan database and dual-luciferase reporter assay verified the interaction between miR-199a-5p and Wnt7b. The results revealed that Wnt7b increased in CNV rats. Knocking down Wnt7b repressed cell proliferation, migration, invasion, and angiogenesis, and accelerated cell apoptosis of HRMEC. Dual-luciferase reporter assay verified that miR-199a-5p targeted Wnt7b. Overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC and promoted cell apoptosis by inhibiting Wbt7b. In vivo experiment found that Wnt7b rescued the promotion of miR-199a-5p inhibition on CNV lesion of rats. In addition, Wnt7b positively regulated Wnt/β-catenin signaling pathway and promoted the angiogenesis of HRMEC. In conclusion, overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC by regulating Wnt7b/Wnt/β-catenin signaling pathway, which may serve as a promising therapy target of CNV.

Cell-Free Culture Supernatant of Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 Reduces the Pathogenicity of NetB-Positive Clostridium perfringens in a Chicken Intestinal Epithelial Cell Line.

The worldwide reduction in the use of antibiotics in animal feed is fueling the need for alternatives for the prevention and control of poultry intestinal diseases such as necrotic enteritis (NE), which is caused by Clostridium perfringens. This is the first report on the use of an intestinal epithelial chicken cell line (CHIC-8E11) to study the pathogenic traits of C. perfringens and to investigate the mode of action of cell-free supernatants (CFS) from probiotic Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 in reducing the pathogenicity of C. perfringens. The cell adhesion, permeability and cytotoxicity were assessed under challenge with four C. perfringens strains isolated from broiler NE episodes of differing geographical origin (CP1-UK; CP10-Sweden; 25037-CP01 and CP22-USA). All the C. perfringens strains could adhere to the CHIC-8E11 cells, with varying affinity (0.05-0.48% adhesion across the strains). The CFS from one out of two strains (CP22) increased the cell permeability (+4.5-fold vs. the control, p < 0.01), as measured by the fluorescein isothiocyanate-dextran (FD4) content, with NetB toxin implicated in this effect. The CFS from all the strains was cytotoxic against the CHIC-8E11 cells in a dose- and strain-dependent manner (cytotoxicity 23-62% across the strains when dosed at 50 µL/mL, as assessed by the MTT cell viability assay). Pre-treatment of the cells with CFS from B. animalis subsp. lactis AG02 but not L. acidophilus AG01 reduced the cell adhesion of three out of four C. perfringens strains (by 77-85% vs. the control, p < 0.001) and reduced the negative effect of two NetB-positive strains on the cell permeability. The CFS of both probiotics alleviated the cytotoxicity of all the C. perfringens strains, which was dependent on the dose. The results confirm the suitability of the CHIC-8E11 cell line for the study of host-pathogen cell interactions in the context of NE caused by C. perfringens and reveal a beneficial mode of action of B. animalis subsp. lactis AG02 in reducing C. perfringens cell adhesion and, together with L. acidophilus AG01, in reducing C. perfringens cytotoxicity.

Oral Microbially-Induced Small Extracellular Vesicles Cross the Blood-Brain Barrier.

Porphyromonas gingivalis (Pg) and its gingipain proteases contribute to Alzheimer's disease (AD) pathogenesis through yet unclear mechanisms. Cellular secretion of small extracellular vesicles or exosomes (EXO) increases with aging as part of the senescence-associated secretory phenotype (SASP). We have shown that EXO isolated from Pg-infected dendritic cells contain gingipains and other Pg antigens and transmit senescence to bystander gingival cells, inducing alveolar bone loss in mice in vivo. Here, EXO were isolated from the gingiva of mice and humans with/without periodontitis (PD) to determine their ability to penetrate the blood-brain barrier (BBB) in vitro and in vivo. PD was induced by Pg oral gavage for 6 weeks in C57B6 mice. EXO isolated from the gingiva or brain of donor Pg-infected (PD EXO) or control animals (Con EXO) were characterized by NTA, Western blot, and TEM. Gingival PD EXO or Con EXO were labeled and injected into the gingiva of uninfected WT mouse model. EXO biodistribution in brains was tracked by an in vivo imaging system (IVIS) and confocal microscopy. The effect of human PD EXO on BBB integrity and permeability was examined using TEER and FITC dextran assays in a human in vitro 3D model of the BBB. Pg antigens (RGP and Mfa-1) were detected in EXO derived from gingival and brain tissues of donor Pg-infected mice. Orally injected PD EXO from donor mice penetrated the brains of recipient uninfected mice and colocalized with hippocampal microglial cells. IL-1β and IL-6 were expressed in human PD EXO and not in Con EXO. Human PD EXO promoted BBB permeability and penetrated the BBB in vitro. This is the first demonstration that microbial-induced EXO in the oral cavity can disseminate, cross the BBB, and may contribute to AD pathogenesis.

Effect of the dietary supplement PERMEAPROTECT+ TOLERANCE© on gut permeability in a human co-culture epithelial and immune cells model

Background and objectiveThe leaky gut syndrome is characterized by an intestinal hyperpermeability observed in multiple chronic disorders. Alterations of the gut barrier are associated with translocation of bacterial components increasing inflammation, oxidative stress and eventually dysfunctions of cellular interactions at the origin pathologies. Therapeutic and/or preventive approaches have to focus on the identification of novel targets to improve gut homeostasis. In this context, this study aims to identify the role of PERMEAPROTECT + TOLERANE©, known as PERMEA, a food complement composed of a combination of factors (including l-Glutamine) known to improve gut physiology. MethodsWe tested the effects of PERMEA or l-Glutamine alone (as reference) on gut permeability (FITC dextran method, expression of tight junctions) and its inflammatory/oxidative consequences (cytokines and redox assays, RT-qPCR) in a co-culture of human cells (peripheral blood mononuclear cells and intestinal epithelial cells) challenged with TNFα. ResultsPERMEA prevented intestinal hyperpermeability induced by inflammation. This was linked with its antioxidant and immunomodulatory properties showing a better efficacity than l-Glutamine alone on several parameters including permeability, global antioxidant charge and production of cytokines. ConclusionPERMEA is more efficient to restore intestinal physiology, reinforcing the concept that combination of food constituents could be used to prevent the development of numerous diseases.

GnT-V-mediated aberrant N-glycosylation of TIMP-1 promotes diabetic retinopathy progression

BackgroundVascular endothelial growth factor (VEGF) signaling pathway plays an important role in the progression of diabetic retinopathy (DR). The glycosylation modification process of many key functional proteins in DR patients is abnormal. However, the potential involvement of abnormal N-glycoproteins in DR progression remains unclear.MethodsGlycoproteomic profiling of the vitreous humor was performed. The level of protein and N-glycoprotein was confirmed by Western blot and Lectin blot, respectively. The cell viability and migration efficiency were detected by CCK-8 and Transwell assay. Flow cytometry was conducted to analyze the level of cell apoptosis and reactive oxygen specie. Malondialdehyde, superoxide dismutase activity and VEGF content were detected by Enzyme linked immunosorbent assays. The interaction of metalloproteinase 1 (TIMP-1) with N-acetylglucosamine transferase V (GnT-V) was detected by GST pull-down. Hematoxylin and eosin staining and choroidal and retinal flat mount stained with fluorescein isothiocyanate-Dextran assay were used for functional research in vivo.ResultsWe found that N-glycosylation was up-regulated in DR rats and high glucose (HG)-induced human retinal pigment epithelium cell line ARPE-19. HG-induced inhibited the viability of ARPE-19 cells and promoted cell apoptosis and oxidative stress (OS), but these effects were reversed with kifunensine treatment, GnT-V knockdown and TIMP-1 mutation. Additionally, GnT-V binds to TIMP-1 to promote N-glycosylation of TIMP-1. Over-expression of GnT-V inhibited the viability of ARPE-19 cells and promoted cell apoptosis, OS and VEGF release, which these effects were reversed with TIMP-1 mutation. Interestingly, over-expression of GnT-V promoted retinal microvascular endothelial cells (RMECs) angiogenesis but was revered with TIMP-1 mutation, which was terminally boosted by VEGF-A treatment. Finally, knockdown of GnT-V relieved DR progression.ConclusionThe findings indicate that GnT-V can promote RMECs angiogenesis and ARPE-19 cells injury through activation VEGF signaling pathway by increasing TIMP-1 N-glycosylation level, which provides a new theoretical basis for the prevention of DR.

Alkaline sphingomyelinase deficiency impairs intestinal mucosal barrier integrity and reduces antioxidant capacity in dextran sulfate sodium-induced colitis.

Ulcerative colitis is a chronic inflammatory disease of the colon with an unknown etiology. Alkaline sphingomyelinase (alk-SMase) is specifically expressed by intestinal epithelial cells, and has been reported to play an anti-inflammatory role. However, the underlying mechanism is still unclear. To explore the mechanism of alk-SMase anti-inflammatory effects on intestinal barrier function and oxidative stress in dextran sulfate sodium (DSS)-induced colitis. Mice were administered 3% DSS drinking water, and disease activity index was determined to evaluate the status of colitis. Intestinal permeability was evaluated by gavage administration of fluorescein isothiocyanate dextran, and bacterial translocation was evaluated by measuring serum lipopolysaccharide. Intestinal epithelial cell ultrastructure was observed by electron microscopy. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction were used to detect the expression of intestinal barrier proteins and mRNA, respectively. Serum oxidant and antioxidant marker levels were analyzed using commercial kits to assess oxidative stress levels. Compared to wild-type (WT) mice, inflammation and intestinal permeability in alk-SMase knockout (KO) mice were more severe beginning 4 d after DSS induction. The mRNA and protein levels of intestinal barrier proteins, including zonula occludens-1, occludin, claudin-3, claudin-5, claudin-8, mucin 2, and secretory immunoglobulin A, were significantly reduced on 4 d after DSS treatment. Ultrastructural observations revealed progressive damage to the tight junctions of intestinal epithelial cells. Furthermore, by day 4, mitochondria appeared swollen and degenerated. Additionally, compared to WT mice, serum malondialdehyde levels in KO mice were higher, and the antioxidant capacity was significantly lower. The expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in the colonic mucosal tissue of KO mice was significantly decreased after DSS treatment. mRNA levels of Nrf2-regulated downstream antioxidant enzymes were also decreased. Finally, colitis in KO mice could be effectively relieved by the injection of tertiary butylhydroquinone, which is an Nrf2 activator. Alk-SMase regulates the stability of the intestinal mucosal barrier and enhances antioxidant activity through the Nrf2 signaling pathway.

Ameliorative Effects of Larazotide Acetate on Intestinal Permeability and Bacterial Translocation in Acute Pancreatitis Model in Rats.

Intestinal barrier dysfunction in acute pancreatitis (AP) may progress to systemic inflammatory response syndrome (SIRS) and multi-organ failures by causing bacterial translocation. Larazotide acetate (LA) is a molecule that acts as a tight junction (TJ) regulator by blocking zonulin (Zo) receptors in the intestine. In our study, we aimed to investigate the effects of LA on intestinal barrier dysfunction and bacterial translocation in the AP model in rats. Thirty-two male Sprague-Dawley rats were divided into 4 groups; control, larazotide (LAR), AP, and AP + LAR. The AP model was created by administering 250 mg/100 g bm L-Arginine intraperitoneally 2 times with an hour interval. AP + LAR group received prophylactic 0.01 mg/mL LA orally for 7 days before the first dose of L-Arginine. For intestinal permeability analysis, fluorescein isothiocyanate-dextran (FITC-Dextran) was applied to rats by gavage. The positivity of any of the liver, small intestine mesentery, and spleen cultures were defined as bacterial translocation. Histopathologically damage and zonulin immunoreactivity in the intestine were investigated. Compared to the control group, the intestinal damage scores, anti-Zo-1 immunoreactivity H-Score, serum FITC-Dextran levels and bacterial translocation frequency (100% versus 0%) in the AP group were significantly higher (all p < 0.01). Intestinal damage scores, anti-Zo-1 immunoreactivity H-score, serum FITC-Dextran levels, and bacterial translocation frequency (50% versus 100%) were significantly lower in the AP + LAR group compared to the AP group (all p < 0.01). Our findings show that LA reduces the increased intestinal permeability and intestinal damage by its effect on Zo in the AP model in rats, and decreases the frequency of bacterial translocation as a result of these positive effects.

Assessment of the Impact of Humic Acids on Intestinal Microbiota, Gut Integrity, Ileum Morphometry, and Cellular Immunity of Turkey Poults Fed an Aflatoxin B1-Contaminated Diet.

A recent study published data on the growth performance, relative weights of the organs of the gastrointestinal tract, liver histology, serum biochemistry, and hematological parameters for turkey poults fed an experimental diet contaminated with aflatoxin B1 (AFB1) and humic acids (HA) extracted from vermicompost. The negative effects of AFB1 (250 ng AFB1/g of feed) were significantly reduced by HA supplementation (0.25% w/w), suggesting that HA might be utilized to ameliorate the negative impact of AFB1 from contaminated diets. The present study shows the results of the remaining variables, as an extension of a previously published work which aimed to evaluate the impact of HA on the intestinal microbiota, gut integrity, ileum morphometry, and cellular immunity of turkey poults fed an AFB1-contaminated diet. For this objective, five equal groups of 1-day-old female Nicholas-700 turkey poults were randomly assigned to the following treatments: negative control (basal diet), positive control (basal diet + 250 ng AFB1/g), HA (basal diet + 0.25% HA), HA + AFB1 (basal diet + 0.25% HA + 250 ng AFB1/g), and Zeolite (basal diet + 0.25% zeolite + 250 ng AFB1/g). In the experiment, seven replicates of ten poults each were used per treatment (n = 70). In general, HA supplementation with or without the presence of AFB1 showed a significant increase (p < 0.05) in the number of beneficial butyric acid producers, ileum villi height, and ileum total area, and a significant reduction in serum levels of fluorescein isothiocyanate-dextran (FITC-d), a marker of intestinal integrity. In contrast, poults fed with AFB1 showed a significant increase in Proteobacteria and lower numbers of beneficial bacteria, clearly suggesting gut dysbacteriosis. Moreover, poults supplemented with AFB1 displayed the lowest morphometric parameters and the highest intestinal permeability. Furthermore, poults in the negative and positive control treatments had the lowest cutaneous basophil hypersensitivity response. These findings suggest that HA supplementation enhanced intestinal integrity (shape and permeability), cellular immune response, and healthier gut microbiota composition, even in the presence of dietary exposure to AFB1. These results complement those of the previously published study, suggesting that HA may be a viable dietary intervention to improve gut health and immunity in turkey poults during aflatoxicosis.

Low dose cadmium inhibits syndecan-4 expression in glycocalyx of glomerular endothelial cells.

Cadmium (Cd) is one of the most polluting heavy metal in the environment. Cd exposure has been elucidated to cause dysfunction of the glomerular filtration barrier (GFB). However, the underlying mechanism remains unclear. C57BL/6J male mice were administered with 2.28 mg/kg cadmium chloride (CdCl2 ) dissolved in distilled water by oral gavage for 14 days. The expression of SDC4 in the kidney tissues was detected. Human renal glomerular endothelial cells (HRGECs) were exposed to varying concentrations of CdCl2 for 24 h. The mRNA levels of SDC4, along with matrix metalloproteinase (MMP)-2 and 9, were analyzed by quantitative PCR. Additionally, the protein expression levels of SDC4, MMP-2/9, and both total and phosphorylated forms of Smad2/3 (P-Smad2/3) were detected by western blot. The extravasation rate of fluorescein isothiocyanate-dextran through the Transwell was used to evaluate the permeability of HRGECs. SB431542 was used as an inhibitor of transforming growth factor (TGF)-β signaling pathway to further investigate the role of TGF-β. Cd reduced SDC4 expression in both mouse kidney tissues and HRGECs. In addition, Cd exposure increased permeability and upregulated P-Smad2/3 levels in HRGECs. SB431542 treatment inhibited the phosphorylation of Smad2/3, Cd-induced SDC4 downregulation, and hyperpermeability. MMP-2/9 levels increased by Cd exposure was also blocked by SB431542, demonstrating the involvement of TGF-β/Smad pathway in low-dose Cd-induced SDC4 reduction in HRGECs. Given that SDC4 is an essential component of glycocalyx, protection or repair of endothelial glycocalyx is a potential strategy for preventing or treating kidney diseases associated with environmental Cd exposure.

A191 THE NEURAL SIGNAL, CALCITONIN GENE-RELATED PEPTIDE (CGRG) ENHANCES A REGULATORY PHENOTYPE IN THE HUMAN IL-4-TREATED MACROPHAGE

Abstract Background Interlukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect in a murine model. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn’s disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, raising the issue of neural modulation of the M(IL4)s reparative function. Aims To determine if CGRP-RAMP1 signalling in human IL-4 treated macrophages enhances the anti-colitic phenotype. Methods Peripheral blood mononuclear cells from healthy volunteers (HD) and individuals with Crohn’s disease were cultured on plastic (2h, 37°C) and non-adherent cells removed. The adherent cells were cultured with recombinant hM-CSF (10 ng/ml) for 7 days. The resultant macrophages (2.5×105) were differentiated with IL-4 (48h 10 ng/mL). In other cells, CGRP (10 nM) was added 24h after IL-4 and phenotype assessed by qPCR. Rag1-/- mice were treated with M(IL4)±CGRP (1x106 i.p.): 48h later, colitis was induced by oxazolone. FITC dextran was used to evaluate epithelial barrier integrity. A wound healing assay was conducted using CaCo2 epithelial cells and conditioned medium (CM,1:2 dilution) from the different treatments. Prostaglandin D2 production was evaluated by ELISA and cyclooxygenase (COX)-1 and -2 activity inhibited using SC560 and indomethacin. Results Compared to non-treated macrophages (M(0)), M(IL4)s from HD had increased RAMP1 and CLR mRNA. M(IL4)s treated with CGRP showed enhanced expression of CD206, CCL26, RAMP1 and CCL18. When delivered systemically to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same healthy blood donor. Use of the the human colon CaCo2 cell in vitro wounding model revealed that CM from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM. Moreover, M(IL4,CGRP)s displayed increased COX-1 and PGD2. CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers. Conclusions Neuronal input is revealed as an important local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer to treat enteric inflammation. Funding Agencies CCCHelmsley Charity

Acute gastrointestinal permeability after traumatic brain injury in mice precedes a bloom in Akkermansia muciniphila supported by intestinal hypoxia

Traumatic brain injury (TBI) increases gastrointestinal morbidity and associated mortality. Clinical and preclinical studies implicate gut dysbiosis as a consequence of TBI and an amplifier of brain damage. However, little is known about the association of gut dysbiosis with structural and functional changes of the gastrointestinal tract after an isolated TBI. To assess gastrointestinal dysfunction, mice received a controlled cortical impact or sham brain injury and intestinal permeability was assessed at 4 h, 8 h, 1 d, and 3 d after injury by oral administration of 4 kDa FITC Dextran prior to euthanasia. Quantification of serum fluorescence revealed an acute, short-lived increase in permeability 4 h after TBI. Despite transient intestinal dysfunction, no overt morphological changes were evident in the ileum or colon across timepoints from 4 h to 4 wks post-injury. To elucidate the timeline of microbiome changes after TBI, 16 s gene sequencing was performed on DNA extracted from fecal samples collected prior to and over the first month after TBI. Differential abundance analysis revealed that the phylum Verrucomicrobiota was increased at 1, 2, and 3 d after TBI. The Verrucomicrobiota species was identified by qPCR as Akkermansia muciniphila, an obligate anaerobe that resides in the intestinal mucus bilayer and produces short chain fatty acids (e.g. butyrate) utilized by intestinal epithelial cells. We postulated that TBI promotes intestinal changes favorable for the bloom of A. muciniphila. Consistent with this premise, the relative area of mucus-producing goblet cells in the medial colon was significantly increased at 1 d after injury, while colon hypoxia was significantly increased at 3 d. Our findings reveal acute gastrointestinal functional changes coupled with an increase of beneficial bacteria suggesting a potential compensatory response to systemic stress after TBI.

Ingestion of Bean Leaves Reduces Metabolic Complications and Restores Intestinal Integrity in C57BL/6 Mice with Obesity Induced by a High-Fat and High-Fructose Diet.

Consumption of foods with fiber and compounds can promote gastrointestinal health and reduce obesity complications. Therefore, treatment with common bean leaves (BL) against obesity was evaluated in mice with a high-fat and high-fructose diet (HFFD) for 14 weeks. The bromatological and phytochemical characterization of BL were determined. Afterwards, the animals were supplemented with BL (10%) or a standard diet (SD) as a strategy to encourage a healthy diet for 12 additional weeks. Changes in body composition, lipid profile, and intestinal integrity were analyzed. The characterization of BL stood out for its content of 27.2% dietary fiber, total phenolics (475.04 mg/100 g), and saponins (2.2 mg/100 g). The visceral adipose tissue (VAT) decreased in the BL group by 52% compared to the HFFD group. Additionally, triglyceride levels were 23% lower in the BL consumption group compared to the HFFD group. The improvement in lipid profile was attributed to the 1.77-fold higher fecal lipid excretion in the BL consumption group compared to the HFFD group and the inhibition of pancreatic lipase by 29%. Furthermore, BL supplementation reduced the serum levels of IL-6 (4.4-fold) and FITC-dextran by 50% compared with those in the HFFD group. Metabolic endotoxemia was inhibited after BL supplementation (-33%) compared to the HFFD group. BL consumption as a treatment in obese mice reduces adipose tissue accumulation and improves the lipid profile. Furthermore, we report for the first time that BL consumption improves intestinal integrity.

EVALUATION OF THE CLINICAL STAGE FXR AGONIST FXR314 IN HUMAN PRIMARY CELL 3D MODELS OF CROHN’S DISEASE AND ULCERATIVE COLITIS

Abstract There is a critical need for novel approaches to treat inflammatory bowel disease (IBD) that extend beyond modulating the immune response. These innovative strategies should also focus on enhancing the integrity of the intestinal barrier and addressing fibrotic tissue development throughout the course of the disease, both critical components of IBD, to improve long term outcomes. Targeting the ligand-activated nuclear Farnesoid X Receptor (FXR) represents such an emerging opportunity. Intestinal epithelial cells exhibit high expression of FXR which is pivotal for intestinal function by regulating the innate immune response, reducing bacterial translocation into the intestinal wall, preserving the integrity of the epithelial barrier, and potentially reducing fibrotic deposition. We have developed a human 3D co-culture model of multiple cell types derived from Crohn’s disease (CD) or ulcerative colitis patients (UC). These 3D-culture CD and UC models faithfully replicate various aspects of the IBD disease state, including differences in gene expression patterns relative to intestinal tissues from healthy donors, compromised epithelial barrier integrity (as measured by Fluorescein isothiocyanate-dextran and TEER assays) and disrupted fibrotic deposition (as determined via fibrosis biomarkers and quantitative collagen deposition). FXR314 is an orally administered non-bile acid FXR agonist that has completed a Phase 2a clinical trial in NASH and is being considered as a novel therapeutic approach for IBD. We assessed the capacity of FXR314 to improve disease biomarkers in 3D models of human IBD. Specifically, its effects were characterized in five CD donors and three UC donors. Target engagement was demonstrated in all CD and UC donors tested through activation of FGF19, a well-accepted target of FXR activation. In CD donors, FXR314 broadly improved measures of epithelial barrier function in a subset of donors, and fibrotic markers in all five donors. In UC donors, FXR314 improved epithelial barrier function and fibrotic activity in all three donors. As anticipated, the effects seen across the different metrics were concentration dependent. These primary multicellular 3D human IBD models (CD, UC) constitute a powerful tool to more accurately predict the relevance of specific targets and assess the efficacy of therapeutic candidates. Consequently, this could enhance the probability of success when conducting clinical trials. In this study, we demonstrated that the non-bile acid FXR agonist FXR314 improves multiple aspects of Crohn’s disease and ulcerative colitis in human donors, thereby strengthening its potential as a novel therapeutic approach for IBD.

Interleukin 17A Contributes to Blood-Brain Barrier Disruption of Hypothalamic Paraventricular Nucleus in Rats With Myocardial Infarction.

Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF. Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN. Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

Ligilactobacillus animalis 506 Protects the Intestinal Barrier from the Damaging Effects of Enteric Pathogens and Deoxynivalenol.

This study investigated the impact of L. animalis 506 on gut barrier integrity and regulation of inflammation in vitro using intestinal epithelial cell lines. Caco-2 or HT29 cell monolayers were challenged with enterotoxigenic E. coli (ETEC) or a ruminant isolate of Salmonella Heidelberg in the presence or absence of one of six probiotic Lactobacillus spp. strains. Among these, L. animalis 506 excelled at exerting protective effects by significantly mitigating the decreased transepithelial electrical resistance (TEER) as assessed using area under the curve (AUC) (p < 0.0001) and increased apical-to-basolateral fluorescein isothiocyanate (FITC) dextran translocation (p < 0.0001) across Caco-2 cell monolayers caused by S. Heidelberg or ETEC, respectively. Similarly, L. animalis 506 and other probiotic strains significantly attenuated the S. Heidelberg- and ETEC-induced increase in IL-8 from HT29 cells (p < 0.0001). Moreover, L. animalis 506 significantly counteracted the TEER decrease (p < 0.0001) and FITC dextran translocation (p < 0.0001) upon challenge with Clostridium perfringens. Finally, L. animalis 506 significantly attenuated DON-induced TEER decrease (p < 0.01) and FITC dextran translocation (p < 0.05) and mitigated occludin and zona occludens (ZO)-1 redistribution in Caco-2 cells caused by the mycotoxin. Collectively, these results demonstrate the ability of L. animalis 506 to confer protective effects on the intestinal epithelium in vitro upon challenge with enteric pathogens and DON known to be of particular concern in farm animals.

Particle Engineering via Supramolecular Assembly of Macroscopic Hydrophobic Building Blocks.

Tailoring the hydrophobicity of supramolecular assembly building blocks enables the fabrication of well-defined functional materials. However, the selection of building blocks used in the assembly of metal-phenolic networks (MPNs), an emerging supramolecular assembly platform for particle engineering, has been essentially limited to hydrophilic molecules. Herein, we synthesized and applied biscatechol-functionalized hydrophobic polymers (poly(methyl acrylate) (PMA) and poly(butyl acrylate) (PBA)) as building blocks to engineer MPN particle systems (particles and capsules). Our method allowed control over the shell thickness (e.g., between 10 and 21 nm), stiffness (e.g., from 10 to 126 mN m-1 ), and permeability (e.g., 28-72 % capsules were permeable to 500 kDa fluorescein isothiocyanate-dextran) of the MPN capsules by selection of the hydrophobic polymer building blocks (PMA or PBA) and by controlling the polymer concentration in the MPN assembly solution (0.25-2.0 mM) without additional/engineered assembly processes. Molecular dynamics simulations provided insights into the structural states of the hydrophobic building blocks during assembly and mechanism of film formation. Furthermore, the hydrophobic MPNs facilitated the preparation of fluorescent-labeled and bioactive capsules through postfunctionalization and also particle-cell association engineering by controlling the hydrophobicity of the building blocks. Engineering MPN particle systems via building block hydrophobicity is expected to expand their use.

Magneto-Mechanical Actuation Induces Endothelial Permeability.

Cancer treatment is one of the major health problems that burden our society. According to the American Cancer Society, over 1.9 million new cancer cases and ∼0.6 million deaths from cancer are expected in the US in 2023. Therapeutic targeting is considered to be the gold standard in cancer treatment. However, when a tumor grows beyond a critical size, its vascular system differentiates abnormally and erratically, creating a heterogeneous endothelial barrier that further restricts drug delivery into tumors. While several methods exist, these prompt tumor migration and the appearance of new metastatic sites. Herein, we propose an innovative method based on magneto-mechanical actuation (MMA) to induce endothelial permeability. This method employs FDA-approved PEGylated superparamagnetic iron oxide nanoparticles (PEG-SPIONs) and alternating nonheating magnetic fields. MMA lies in the translation of magnetic forces into mechanical agitation. As a proof of concept, we developed a 2D cell culture model based on human umbilical vein endothelial cells (HUVEC), which were incubated with PEG-SPIONs and then exposed to different magnetic doses. After adjusting the particle concentration, incubation times, and parameters (amplitude, frequency, and exposure time) of the magnetic field generator, we induced actin filament remodeling and subsequent vascular endothelial-cadherin junction disruption. This led to transient gaps in cell monolayers, through which fluorescein isothiocyanate-dextran was translocated. We observed no cell viability reduction for 3 h of particle incubation up to a concentration of 100 μg/mL in the presence and absence of magnetic fields. For optimal permeability studies, the magnetic field parameters were adjusted to 100 mT, 65 Hz, and 30 min in a pulse mode with 5 min OFF intervals. We found that the endothelial permeability reached the highest value (33%) when 2 h postmagnetic field treatment was used. To explain these findings, a magneto-mechanical transduced stress mechanism mediated by intracellular forces was proposed. This method can open new avenues for targeted drug delivery into anatomic regions within the body for a broad range of disease interventions.