Human Intestinal Epithelial Caco-2 Cells Research Articles

NPC1L1-dependent transport of 27-alkyne cholesterol in intestinal epithelial cells.

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

ADENOSINE A3 RECEPTOR INTERACTS WITH GASDERMIN D TO MODULATE INTESTINAL EPITHELIAL CELL PYROPTOSIS IN ULCERATIVE COLITIS

Abstract Background Adenosine A3 receptor (A3AR) plays a role in intestinal inflammation, but little is known about its mechnisms in intestinal inflammation such as ulcerative colitis (UC). Pyroptosis, characterized by Gasdermin D (GSDMD) activation, is implicated in the pathogenesis of UC. We investigated the role of A3AR in GSDMD-mediated pyroptosis in UC and its underlying molecular mechanisms. Methods The expression of A3AR in colonic mucosa of patients with UC were examined. A3AR agonist was used to study the role of A3AR in ex vivo colonic explants of UC patients. In addition, human intestinal epithelial cells Caco-2 were used to further verify the effect of A3AR on pyroptosis induced by LPS+ATP. RT-qPCR and western blotting were used to detect the expression levels of pyroptosis-associated factors including NLRP3, caspase-1, gasdermin-D N-terminal domain (GSDMD-NT), IL-1β and IL-18 in colonic tissues and Caco-2 cells. Immunofluorescence was used to detect the protein expression in tissues and cells. Enzyme-linked immunosorbent assay was used to determine the levels of IL-1β and IL-18 in tissue and cell culture supernatants. Lactate dehydrogenase (LDH) release assay and propidium iodide (PI) staining were used to measure cell pyroptosis. Molecular interactions beween A3AR and GSDMD were investigated using Co-IP and GST pull-down assays. Results A3AR expression was significantly reduced in colonic mucosa of patients with active UC, and colonic epithelial cells were the main cell subpopulation with down-regulated A3AR expression. Pyroptosis-associated factors, including Caspase-1, NLRP3, and GSDMD-NT, were upregulated in UC colonic tissues. The expression of A3AR and GSDMD-NT was negatively correlated. A3AR agonist reduced the production of cytokines (IL-1β and IL-18) and attenuates the expression levels of NLRP3 and GSDMD-NT in the colonic tissues of patients with UC. Furthermore, A3AR overexpression alleviated pyroptosis with reduced LDH release, PI-stained cell number and decreased expressions of GSDMD-NT, NLRP3, caspase-1, IL-1β, and IL-18 in the LPS+ATP-stimulated Caco-2 cells, whereas the opposite occurred in cells treated with small interfeing RNA (siRNA) targeting A3AR. Knockdown of GSDMD in Caco-2 cells significantly blocked the effects of A3AR overexpression or down-regulation on pyroptosis, suggesting that A3AR acts through the GSDMD-mediated pyroptosis pathway. Co-IP and GST pull-down assays showed that A3AR interacted with GSDMD. Conclusion A3AR modulates intestinal inflammation in UC through GSDMD-mediated intestinal epithelial cell pyroptosis. We propose a novel mechanism by which A3AR-GSDMD interaction affects UC through pyroptosis, suggesting that A3AR is a potential target for the treatment of UC.

Anemoside B4 ameliorates TNBS-induced colitis through S100A9/MAPK/NF-\u03baB signaling pathway

BackgroundDespite the increased morbidity of ulcerative colitis (UC) in the developing countries, available treatments remain unsatisfactory. Therefore, it is urgent to discover more effective therapeutic strategies. Pulsatilla chinensis was widely used for the treatment of inflamed intestinal diseases including UC for thousands of years in China. Anemoside B4, the most abundant triterpenoid saponin isolated from P. chinensis, exerts anti-inflammatory and antioxidant effects and may be the most active compounds, which is responsible for the therapeutic effects. However, the mechanism how anemoside B4 executes its biological functions is still elusive.MethodsHere, we used the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rat model to evaluate the therapeutic effect of anemoside B4. Blood samples of colitis rats were collected for hematology analysis. The inflammation-associated factors were investigated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis was determined with EdU cell proliferation assay and TUNEL assay. The proteins regulated by anemoside B4 were identified by label-free quantitative proteomics. The significantly down-regulated proteins were verified by Western blotting analysis. mRNA expression was analyzed by quantitative real-time RT-PCR.ResultsThe results showed that anemoside B4 ameliorated TNBS-induced colitis symptoms, including tissue damage, inflammatory cell infiltration, and pro-inflammatory cytokine production, apoptosis and slowed proliferation in colon. Quantitative proteomic analyses discovered that 56 proteins were significantly altered by anemoside B4 in the TNBS-induced rats. These proteins mainly clustered in tricarboxylic acid (TCA) cycle and respiratory electron transport chain. Among the altered proteins, S100A9 is one of the most significantly down-regulated proteins and associated with NF-κB and MAPK signaling pathways in the pathogenesis of UC. Further experiments revealed that anemoside B4 suppressed the expression of S100A9 and its downstream genes including TLR4 and NF-κB in colon. In vitro, anemoside B4 could inhibit the NF-κB signaling pathway induced by recombinant S100A9 protein in human intestinal epithelial Caco-2 cells. Moreover, anemoside B4 inhibits neutrophils recruitment and activation in colon induced by TNBS.ConclusionsOur results demonstrate that anemoside B4 prevents TNBS-induced colitis by inhibiting the NF-κB signaling pathway through deactivating S100A9, suggesting that anemoside B4 is a promising therapeutic candidate for colitis.

Impact of soy lecithin, zinc oxide, and methylsulfonylmethane, as excipient ingredients, on the bioaccessibility and intestinal transport of branched-chain amino acids from animal and plant protein mixtures.

To maximize the biological activity of branched-chain amino acids (BCAAs), it is necessary to find a new excipient agent to increase the bioavailability of BCAAs in protein mixtures. The aim of the current study was to investigate the effects of soy lecithin (SLC), zinc oxide (ZnO), and methylsulfonylmethane (MSM) on the bioaccessibility and intestinal transport of BCAAs from animal and plant protein mixtures (PMs) via an in vitro digestion model with human intestinal epithelial (Caco-2) cells. The bioaccessibility of total BCAAs in PMs considerably increased by 107.51 ± 1.50% with the addition of SLC, and the combined effects of SLC, ZnO, and MSM on enhancing the bioaccessibility of total BCAAs was observed (107.14 ± 0.18%). Interestingly, SLC showed a major role in binding bile acid, showing 65.78 ± 1.66% of binding capacity. Intestinal transport of BCAAs was measured to be at 100.48, 110.86, and 130.29 μg mL-1 for leucine, isoleucine, and valine, respectively, in PMs with SLC + ZnO + MSM, and it eventually amplified the amount of the total transported BCAAs (341.63 ± 6.34 μg mL-1), which was about 8.72 times higher than that of PM only. The cellular integrity of digesta-treated Caco-2 cells tended to decrease according to the incubation time, but it was recovered in the treatment of PM + SLC + ZnO + MSM, and nearly reached the control levels with 92.82 ± 0.53%. Results from the current study suggest that the co-consumption of proteins equally consisting of plant and animal sources with SLC, ZnO, and MSM could improve the bioavailability of total BCAAs, resulting in the improvement of health benefits.

Vitamin E alpha- and gamma-tocopherol mitigate colitis, protect intestinal barrier function and modulate the gut microbiota in mice

Inflammatory bowel diseases (IBDs) including colitis are intestinal disorders characterized by chronic inflammation, barrier dysfunction and dysbiosis. Specific forms of vitamin E have been shown to attenuate colitis, but the mechanisms are not fully understood. The objective of this study is to examine the impact of α-tocopherol (αT) and γ-tocopherol-rich tocopherols (γTmT) on gut inflammation, barrier integrity and microbiota in dextran sulfate sodium (DSS)-induced colitis in mice. We observe that αT and γTmT mitigated DSS-caused fecal bleeding, diarrhea and elevation of IL-6. These vitamin E forms inhibited colitis-induced loss of the tight junction protein occludin, and attenuated colitis-caused elevation of LPS-binding protein in the plasma, a surrogate marker of intestinal barrier dysfunction, suggesting protection of gut barrier integrity. Consistently, αT and γT mitigated TNF-α/IFN-γ-induced impairment of trans-epithelial electrical resistance in human intestinal epithelial Caco-2 cell monolayer. Using 16S rRNA gene sequencing of fecal DNA, we observe that DSS reduced gut microbial evenness and separated microbial composition from healthy controls. In colitis-induced mice, γTmT but not αT separated gut microbial composition from controls, and attenuated DSS-caused depletion of Roseburia, which contains butyrate producing bacteria and is decreased in IBD patients. Canonical correspondence analysis also supports that γTmT favorably altered gut microbial community. In contrast, neither αT nor γTmT affected gut microbes in healthy animals. These results provide evidence supporting protective effects of αT and γT on intestinal barrier function and that γTmT caused favorable changes of the gut microbiota in colitis-induced mice.

Differences in Toxic Response Induced by Three Variants of the Diarrheic Shellfish Poisoning Phycotoxins in Human Intestinal Epithelial Caco-2 Cells.

Diarrheic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated with a group of phycotoxins that includes okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2). These toxins are inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), but show distinct levels of toxicity. Aside from a difference in protein phosphatases (PP) inhibition potency that would explain these differences in toxicity, others mechanisms of action are thought to be involved. Therefore, we investigated and compared which mechanisms are involved in the toxicity of these three analogues. As the intestine is one of the target organs, we studied the transcriptomic profiles of human intestinal epithelial Caco-2 cells exposed to OA, DTX-1, and DTX-2. The pathways specifically affected by each toxin treatment were further confirmed through the expression of key genes and markers of toxicity. Our results did not identify any distinct biological mechanism for OA and DTX-2. However, only DTX-1 induced up-regulation of the MAPK transduction signalling pathway, and down-regulation of gene products involved in the regulation of DNA repair. As a consequence, based on transcriptomic results, we demonstrated that the higher toxicity of DTX-1 compared to OA and DTX-2 was consistent with certain specific pathways involved in intestinal cell response.

Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

Virulence characterization and comparative genomics of Listeria monocytogenes sequence type 155 strains

BackgroundListeria (L.) monocytogenes strains show a high diversity regarding stress tolerance and virulence potential. Genome studies have mainly focused on specific sequence types (STs) predominantly associated with either food or human listeriosis. This study focused on the prevalent ST155, showing equal distribution among clinical and food isolates. We evaluated the virulence potential of 20 ST155 strains and performed comparative genomic analysis of 130 ST155 strains isolated from food, food processing environments and human listeriosis cases in different countries and years.ResultsThe in vitro virulence assays using human intestinal epithelial Caco2 and hepatocytic HEPG2 cells showed an impaired virulence phenotype for six of the 20 selected ST155 strains. Genome analysis revealed no distinct clustering of strains from the same source category (food, food processing environment, and clinical isolates). All strains harbored an intact inlA and inlB locus, except four strains, which had an internal deletion in the inlA gene. All strains harbored LIPI-1, but prfA was present in a longer variant in six strains, all showing impaired virulence. The longer PrfA variant resulted in lower expression of inlA, inlB, and prfA, and no expression of hly and actA.Regarding stress-related gene content, SSI-1 was present, whereas qacH was absent in all strains. 34.6% of the strains harbored a plasmid. All but one ST155 plasmids showed high conservation and harbored cadA2, bcrABC, and a triphenylmethane reductase.ConclusionsThis study contributes to an enhanced understanding of L. monocytogenes ST155 strains, being equally distributed among isolates from humans, food, and food processing environments. The conservation of the present genetic traits and the absence of unique inherent genetic features makes these types of STs especially interesting since they are apparently equally adapted to the conditions in food processing environments, as well as in food as to the human host environment. However, a ST155-specific mutation resulting in a longer PrfA variant impaired the virulence potential of several ST155 strains.

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.

The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.

Cytotoxic, Genotoxic, and Apoptotic Effects of Nickel Oxide Nanoparticles in Intestinal Epithelial Cells.

The superior properties of nickel oxide-nanoparticles (NiO-NPs) have led to their wide use in various fields. However, there is little comprehensive knowledge about their toxicity, especially after oral exposure. The toxic effect of NiO-NPs of mean size 15.0 nm was investigated in Caco-2 (human intestinal epithelial) cells as no study has been performed on their intestinal toxicity. Following identification of their particle size distribution and cellular uptake potential, the risk of exposure to NiO-NPs was evaluated by cellular morphologic changes, cyto- and genotoxic potentials, oxidative damage, and apoptotic induction. NiO-NPs induced a 50% reduction in cell viability at 351.6 μg/mL and caused DNA damage and oxidative damage at 30-150 μg/mL. It appears that apoptosis might be a main cell death mechanism in NiO-NP-exposed intestinal cells. NiO-NPs might be hazardous to the gastrointestinal system. The results should raise concerns about using NiO-NPs in food-contact appliances and about NiO-NP-containing wastes. Further in vivo and in vitro research should be conducted to explain the specific toxicity mechanism of these particles and reduce their risk to humans.

Involvement of Organic Cation Transporter 2 and a Na+-dependent active transporter in p-tyramine transport across Caco-2 intestinal cells

AimsWe have previously demonstrated that p-tyramine (TYR), an endogenous trace amine-associated receptor 1 agonist, passage across neuronal membranes involves a transporter exhibiting the pharmacological profile of Organic Cation Transporter 2 (OCT2). Since TYR is also a constituent of foodstuffs and produced by the intestinal microbiota, here we have investigated whether similar processes are involved in the passage of 100 nM TYR across apical and basolateral membranes of the Caco-2 human intestinal epithelial cell line. Materials and methods[3H]TYR transport across apical and basolateral membranes of Caco-2 cell monolayers was measured in the presence of inhibitors of TYR metabolizing enzymes. Cellular, apical, and basolateral compartments were collected at various timepoints, TYR concentrations calculated, and transport properties pharmacologically characterized. Key findingsApical transport resulted in equimolar accumulation of TYR within cells. Pentamidine (OCT1/OCT2 inhibitor) decreased apical transport (P = 0.001) while atropine (OCT1 inhibitor) had no effect, suggesting apical transport involved OCT2. In contrast, basolateral transport resulted in 500–1000 nM cellular concentrations (P < 0.0001) indicating the presence of an active transporter. Replacement of Na+ on an equimolar basis with choline resulted in loss of TYR transport (P = 0.017). Unexpectedly, this active transport was also atropine-sensitive (P = 0.020). Kinetic analysis of the active transporter revealed Vmax = 43.0 nM/s with a Kt = 33.1 nM. SignificanceWe have demonstrated for the first time that TYR is transported across Caco-2 apical membranes via facilitated diffusion by OCT2, whereas transport across basolateral membranes is by a Na+-dependent, atropine-sensitive, active transporter.

Chondroitin sulfate and its nanocomposites with protamine or chitosan stabilize and deliver available nanosized iron

Nanostructured iron (III) compounds are promising candidates for iron fortification applications due to their good solubility, bioavailability, and redox inertia. The current study synthesized ferric oxyhydroxide nanoparticles (FeONPs) based on chondroitin sulfate (ChS) and its nanocomposites with protamine sulfate (PS) or chitosan (CS) in neutral aqueous solution under ambient conditions, and evaluated their iron availability to polarized human intestinal epithelial (Caco-2) cells. Ultraviolet-visible spectroscopy, dynamic light scattering, transmission electron microscopy and energy-dispersive X-ray spectroscopy showed that ChS-FeONPs were wave-like anionic particles where FeONPs attached on the polysaccharide chains and PS/ChS-FeONPs and CS/ChS-FeONPs were irregular anionic nanoparticles where FeONPs scattered across the entire region. The calcein-fluorescence-quenching assay in polarized Caco-2 cells showed good iron uptake from ChS-FeONPs, PS/ChS-FeONPs and CS/ChS-FeONPs mainly via endocytosis, with the latter two exhibiting better iron absorption. Overall, our study provides a more facile and greener alternative route to synthesize the bioavailable nano-sized iron.

Suppressive Effects of Hot-water Extract of Magnolia obovata on Clostridium perfringens Enterotoxin-induced Cytotoxicity in Human Intestinal Caco-2 Cells.

The physiological functions of the leaves of Japanese big-leaf magnolia (Magnolia obovata) against enterotoxins produced by foodborne pathogens remain unclear. In this study, we evaluated the protective effects of M.obovata leaf extract (MLE) against the cytotoxicity of Clostridium perfringens enterotoxin (CPE), which causes the symptoms of C. perfringens type A food poisoning. The protective effects of MLE against CPE-induced cytotoxicity were evaluated in human intestinal epithelial Caco-2 cells. Pre-treatment with MLE significantly suppressed the cytotoxicity induced by CPE in undifferentiated and differentiated human intestinal Caco-2 cells at a pH range of 4.0 - 7.0. This CPE-suppressive effect was due to a hydrophilic sugar-containing compound without phenolic and protein structures but not the hydrophobic biologically active neolignans, honokiol and magnolol. MLE had a protective effect against cytotoxicity caused by type A C. perfringens. Our results provide novel insight regarding the usage of M.obovata in managing food poisoning.

Microalgae aqueous extracts exert intestinal protective effects in Caco-2 cells and dextran sodium sulphate-induced mouse colitis.

Microalgae are emerging as a good source of natural nutraceuticals. Here, we examined the intestinal protective effects of microalgae aqueous extracts (MAEs) from Chlorella pyrenoidosa, Spirulina platensis, and Synechococcus sp. PCC 7002 in human intestinal epithelial Caco-2 cells and dextran sodium sulphate (DSS)-induced colitis in C57BL/6 mice. MAEs displayed intestinal barrier-protective activities in Caco-2 cells by increasing the expression of heat shock protein (Hsp)-27 and tight junction proteins of occludin and claudin-4 and attenuating the H2O2-induced intracellular reactive oxygen species production, plasma membrane impairment and apoptosis. They also showed anti-inflammatory potential in tumor necrosis factor (TNF)-α-, interleukin (IL)-1β- and H2O2-stimulated Caco-2 cells by suppressing the secretion of IL-8 and the expression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). The 8 d daily intragastric administration of MAEs during and after 4 d DSS exposure effectively alleviated colitis symptoms of weight loss, diarrhea, rectal bleeding, and colon shortening and histopathology, protected intestinal barrier function by increasing colonic Hsp-25, occludin and claudin-4, and attenuated colonic and systemic inflammation by suppressing colonic myeloperoxidase activity, production of TNF-α, IL-1β and IL-6, expression of COX-2 and iNOS, and peripheral leukocytosis, monocytosis and granulocytosis. Microalgae can thus serve as a functional food to maintain gut health.

Nanoplastics as a potential environmental health factor: effects of polystyrene nanoparticles on human intestinal epithelial Caco-2 cells

Our results show that both nPS are easily internalized by Caco-2 cells in a concentration-dependent manner, but no relevant toxic effects are observed under the analyzed exposure conditions.

Determination and prediction of permeability across intestinal epithelial cell monolayer of a diverse range of industrial chemicals/drugs for estimation of oral absorption as a putative marker of hepatotoxicity

Apparent permeability coefficients (Papp) across a human intestinal epithelial Caco-2 cell monolayer were measured for a range of industrial/drug chemicals. A predictive equation for determining in vitro Papp values of fifty-six substances was set up using multivariate regression analysis based on in silico-estimated physicochemical properties (molecular weights and water distribution coefficients for apical and basal pH environments) (r = 0.77, p < 0.01). Predicted logPapp values of a secondary set of 34 compounds were correlated with the measured values. Under the medicinal logPapp values associated with their reported fraction absorbed, a significant inverse non-linear correlation was found between the logarithmic transformed values of observed Papp values and reported hepatic no-observed-effect levels of industrial chemicals (r = –0.55, p < 0.01, n = 29). In vitro determination and/or in silico prediction of permeability across intestinal cells could be effective for estimating oral absorption as a putative indicator for hepatotoxicity.

PSVI-11 Antioxidative effects of red-osier dogwood extract on H2O2-treated human intestinal epithelial Caco-2 cells

Abstract Red-osier dogwood is a species of flowering plant native in North America. Its extract may exhibit the anti-oxidative and anti-bacterial properties because it contains plenty of phenolic compounds, polysaccharides, and bioactive peptides. Hence, red-osier dogwood extract (RDE) could be a potential alternative to antibiotics. In the present study, an in vitro oxidative damage model via using Caco-2 cells was established to evaluate the antioxidative effects of RDE. The results showed that 1 mM H2O2 significantly decreased the cell viability, and increased interleukin 8 (IL-8) secretion and reactive oxygen species (ROS) production (P &amp;lt; 0.05). The mRNA abundance of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and IL-8 also increased in cells treated by H2O2. Caco-2 cells exposed to H2O2 for 6 h were then treated with 100 µg/mL RDE for 24 h. The IL-8 secretion and ROS production induced by H2O2 was significantly blocked by RDE (P &amp;lt; 0.05). RDE reduced the leakage of Fluorescein isothiocyanate (FITC)-dextran, and increased transepithelial resistance (TEER) value compared with the control (H2O2 treatment). Moreover, RDE increased Zonula occludens-1 (ZO-1) and β-actin staining in the cells and tight junction proteins expression including ZO-1, claudin 1, claudin 3 and occludin. These results suggested that RDE enhanced barrier functions and reduced ROS production in the epithelial cells under H2O2-induced oxidative damage.

Production of functional mimics of human milk oligosaccharides by enzymatic glycosylation of bovine milk oligosaccharides

Consumption of mothers' milk is associated with reduced incidence and severity of enteric infections, leading to reduced morbidity in breastfed infants. Fucosylated and sialylated human milk oligosaccharides (HMO) are important for both direct antimicrobial action – likely via a decoy effect – and indirect antimicrobial action through commensal growth enhancement. Bovine milk oligosaccharides (BMO) are a potential source of HMO-mimics as BMO resemble HMO; however, they have simpler and less fucosylated structures. BMO isolated at large scales from bovine whey permeate were modified by the addition of fucose and/or sialic acid to generate HMO-like glycans using high-yield and cost-effective one-pot multienzyme approaches. Quadrupole time-of-flight LC/MS analysis revealed that 22 oligosaccharides were synthesized and 9 had identical composition to known HMO. Preliminary anti-adherence activity assays indicated that fucosylated BMO decreased the uptake of enterohemorrhagic Escherichia coli O157:H7 by human intestinal epithelial Caco-2 cells more effectively than native BMO.

Impact of exposure to cold and cold-osmotic stresses on virulence-associated characteristics of Listeria monocytogenes strains

The objective of this work was to investigate the effect of stress conditions frequently encountered in food-associated environments on virulence-associated characteristics of eight strains of Listeria monocytogenes. Strains were grown at low (11 °C, cold stress) and optimal (37 °C) temperatures and in high NaCl concentrations (6% NaCl, 11 °C; cold-osmotic stress) and tested for their ability to invade the human intestinal epithelial Caco-2 cells. Results demonstrate that the correlation between exposure to cold stress and increased invasion phenotype is strain-dependent as strains investigated exhibited different behaviours, i.e. exposure to cold stress conditions resulted in a significant increase of invasion levels in five out of the eight strains tested, when compared to growth under optimal conditions. On the other hand, when these cold-adapted cells were subsequently submitted to high salt concentrations and low temperature, their enhanced ability to invade Caco-2 was lost. Surprisingly, saturated fatty acids (SFA) and branched chain fatty acids (BCFA) decreased when L. monocytogenes were exposed to stress conditions as opposed to what has been observed in other studies, therefore highlighting that further studies will need to deepen in the understanding of the lipid metabolism of these strains. The effect of stress conditions on the survival of three selected L. monocytogenes strains through an in vitro gastrointestinal (GI) tract digestion model was further investigated. The exposure to cold-osmotic stress increased the survival of one strain through the GI tract.

Evaluation of Incompatibility Group I1 (IncI1) Plasmid-Containing Salmonella enterica and Assessment of the Plasmids in Bacteriocin Production and Biofilm Development.

Mobile genetic elements, such as plasmids, can potentially increase the ability of bacteria to infect and persist in vertebrate host cells. IncI1 plasmids are widely distributed in Salmonella from food animal sources and associated with clinically important strains. These plasmids often encode antimicrobial resistance; however, little is known about their impact on the virulence of Salmonella strains. To assess the potential impact of the plasmids on virulence, 43 IncI1-positive Salmonella isolates from human and animal sources were subjected to whole genome sequence (WGS) analyses and evaluated for their abilities to invade and persist for 48 h in Caco-2 human intestinal epithelial cells, form biofilms and encode bacteriocins. Draft WGS data were submitted to predict the presence of virulence and antimicrobial resistance genes, plasmid replicon types present, conduct plasmid multilocus sequence typing (pMLST), and core genome MLST (cgMLST) in the isolates. Caco-2 cells were infected with Salmonella strains and incubated for both one and 48 h for the invasion and persistence assays, respectively. Additionally, Salmonella isolates and IncI1 plasmid carrying transconjugants (n = 12) generated in Escherichia coli were assessed for their ability to produce biofilms and bacteriocin inhibition of growth of other bacteria. All Salmonella isolates infected Caco-2 cells and persisted in the cells at 48 hrs. Persistent cell counts were observed to be significantly higher than invasion assay cell counts in 26% of the isolates. Among the IncI1 plasmids, there were 18 pMLST types. Nearly 35% (n = 15) of Salmonella isolates produced biofilms; however, none of the IncI1-positive transconjugants produced increased biofilms compared to the recipient. Approximately 65% (n = 28) of isolates and 67% (n = 8) of IncI1-positive transconjugants were able to inhibit growth of at least one E. coli strain; however, none inhibited the growth of strains from species other than E. coli. The study characterized IncI1 positive Salmonella isolates and provided evidence about the potential contributions of IncI1 plasmids virulence phenotypes and areas where they do not. These findings should allow for more focused efforts to assess the impact of plasmids on bacterial pathophysiology and human health.