Nitric oxide synthase induction and intestinal epithelial cell viability in rats

The present study determined the presence of constitutive and inducible nitric oxide (NO) synthase activities in intestinal isolated epithelial cells and the effects of NO induction on intestinal epithelial cell viability. Epithelial cells were isolated from rat proximal small intestine by dispersion using citrate and EDTA. Constitutive NO synthase activity, determined by [14C]arginine conversion to citrulline that was inhibited by in vitro incubation with the arginine analogue NG-monomethyl-L-arginine (L-NMMA; 300 microM) or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 1 mM), was observed in these cells. Administration of Escherichia coli lipopolysaccharide (LPS; 3 mg/kg iv) significantly augmented NO synthase activity (determined 4 h later), which was inhibited in vitro by incubation with L-NMMA but not by EGTA. The highest level of constitutive and inducible NO synthase activity occurred in intestinal villus cells, and the lowest was in crypt cells. Induction of NO synthase activity was associated with a decrease in cellular viability as assessed by a decrease in trypan blue exclusion. Dexamethasone pretreatment (1 mg/kg iv 2 h before LPS administration) significantly reduced both induction of NO synthase activity and the reduction in cellular viability. Likewise concurrent administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg sc) ameliorated the reduction in cell viability induced by LPS administration, an effect abolished by pretreatment with the NO substrate L-arginine (350 mg/kg sc). Whereas constitutively formed NO may have a physiological role in these cells, the results in this study suggest that induction of NO synthase in epithelial cells may represent a mechanism of local intestinal damage.

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Retraction for Hunter et al., “ Lactobacillus bulgaricus Prevents Intestinal Epithelial Cell Injury Caused by Enterobacter sakazakii -Induced Nitric Oxide both In Vitro and in the Newborn Rat Model of Necrotizing Enterocolitis”

Infection and Immunity ◽

10.1128/iai.00212-18 ◽

2018 ◽

Vol 86 (6) ◽

Author(s):

Catherine J. Hunter ◽

Monica Williams ◽

Mikael Petrosyan ◽

Yigit Guner ◽

Rahul Mittal ◽

...

Keyword(s):

Nitric Oxide ◽

Epithelial Cell ◽

Rat Model ◽

Intestinal Epithelial Cell ◽

Cell Injury ◽

Lactobacillus Bulgaricus ◽

Intestinal Epithelial ◽

Enterobacter Sakazakii ◽

Newborn Rat

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Rapid protein kinase D1 signaling promotes migration of intestinal epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00025.2012 ◽

2012 ◽

Vol 303 (3) ◽

pp. G356-G366 ◽

Cited By ~ 10

Author(s):

Steven H. Young ◽

Nora Rozengurt ◽

James Sinnett-Smith ◽

Enrique Rozengurt

Keyword(s):

Protein Kinase ◽

Epithelial Cell ◽

Epithelial Cells ◽

Transgenic Mice ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Protein Kinase D1

We have examined the role of protein kinase D1 (PKD1) signaling in intestinal epithelial cell migration. Wounding monolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid PKD1 activation in the cells immediately adjacent to the wound edge, as judged by immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD1 at Ser916, an autophosphorylation site. An increase in PKD1 phosphorylation at Ser916 was evident as early as 45 s after wounding, reached a maximum after 3 min, and persisted for ≥15 min. PKD1 autophosphorylation at Ser916 was prevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A kb NB 142-70-sensitive increase in PKD autophosphorylation was also elicited by wounding IEC-6 cells. Using in vitro kinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cells induced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling is required to promote migration of intestinal epithelial cells into the denuded area of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAs targeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. To test whether PKD1 promotes migration of intestinal epithelial cells in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestinal epithelium. Enterocyte migration was markedly increased in the PKD1 transgenic mice. These results demonstrate that PKD1 activation is one of the early events initiated by wounding a monolayer of intestinal epithelial cells and indicate that PKD1 signaling promotes the migration of these cells in vitro and in vivo.

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PSIX-17 Investigating the biological activities of sodium cellobionate produced from cellulosic biomass

Journal of Animal Science ◽

10.1093/jas/skaa054.330 ◽

2020 ◽

Vol 98 (Supplement_3) ◽

pp. 187-187

Author(s):

Lauren Kovanda ◽

Zhiliang Fan ◽

Xunde Li ◽

Yanhong Liu

Keyword(s):

Cell Proliferation ◽

Antimicrobial Activity ◽

Epithelial Cell ◽

Epithelial Cells ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Biological Activities ◽

Radical Scavenging ◽

Intestinal Epithelial

Abstract A novel method has been developed to easily hydrolyze cellulose to sodium cellobionate in a filamentous fungas, Neurospora crassa. The objectives of this experiment were to investigate the in vitro biological activities of sodium cellobionate. Antioxidant activity was evaluated with 3 chemical-based assays, including DPPH radical scavenging assay (DPPH), Trolox equivalent antioxidant capacity assay (TEAC), and ferric reducing antioxidant power assay (FRAP). Antimicrobial activity was determined as minimum inhibitory concentration (MIC) that prevented growth of tested bacteria, including four gram-negative bacteria (Escherichia coli F18 and ATCC 25922, and Salmonella Typhimurium ATCC 14028 and a wild strain isolated from cull diary cows in California) and one gram-positive bacteria (Enterococcus faecalis ATCC 29212). Anti-inflammatory activity was tested by analyzing TNF-α production with porcine alveolar macrophages that were challenged with lipopolysaccharide. A porcine intestinal epithelial cell line, IPEC-J2, was also used to test the effects of cellobionate on cell proliferation of intestinal epithelial cells. The tested doses of sodium cellobionate were 0, 0.04, 0.20, 1.00, 2.00, 4.00, 20.00, and 40.00 mg/mL. All assays were performed with over 6 replicates, except that MIC assays were performed as triplicate. All data were analyzed by PROC MIXED of SAS. Sodium cellobionate did not have radical scavenging capacity, but had weak FRAP (9.68 μM L-Cysteine equivalent) and TEAC (69% reduction) at the dose of 40 mg/mL. MIC results revealed that sodium cellobionate did not inhibit the growth of all tested bacteria, indicating it does not have antimicrobial activity within the range of tested doses. Sodium cellobionate did not exhibit anti-inflammatory activities, but significantly enhanced (P < 0.05) intestinal epithelial cell proliferation in vitro by 24.00%, 39.64%, and 25.98% when the doses were 1.00, 2.00, and 4.00 mg/mL, respectively. Results of this experiment indicate that cellobionate has limited biological activities in vitro, except that this biomass product could strongly stimulate the proliferation of intestinal epithelial cells. Future research will focus on the potential impacts of sodium cellobionate on intestinal physiology in vivo.

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62 Evaluating detoxification efficacy of deoxynivalenol by encapsulated sodium metabisulfite (SMBS) using an in vitro intestinal epithelial cell IPEC-J2 model

Journal of Animal Science ◽

10.1093/jas/skaa278.069 ◽

2020 ◽

Vol 98 (Supplement_4) ◽

pp. 38-39

Author(s):

Peng Lu ◽

Changning Yu ◽

Shangxi Liu ◽

Joshua Gong ◽

Song Liu ◽

...

Keyword(s):

Small Intestine ◽

Epithelial Cell ◽

Epithelial Cells ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

In Vitro Release ◽

Simulated Gastric Fluid ◽

Sodium Metabisulfite ◽

Intestinal Epithelial

Abstract Deoxynivalenol (DON) contamination occurs on feed ingredients and causes a reduction in growth performance, damage to the intestinal epithelial cells, and increased susceptibility to enteric pathogen challenge. Sodium metabisulfite (SMBS) has been successfully used to destroy DON in processed grains or feeds. However, SMBS degrades quickly under aqueous acid conditions, such as pig stomachs, and when SMBS is added to diet, little will remain intact in the small intestine where an optimal pH environment exists for detoxification by SMBS. Thus, this study was to encapsulate SMBS into microparticles to deliver intact SMBS to the small intestine and evaluate its efficacy of DON detoxification in the simulated intestine fluid (SIF) using an in vitro intestinal epithelial cell (IPEC-J2) model. The results showed that around 40% of the SMBS loading capacity was achieved in the microparticles. In vitro release studies showed that 1.61% of encapsulated SMBS was released in the simulated gastric fluid (SGF), and the majority of encapsulated SMBS (75.52%) was progressively released in the SIF within 6 h at 37 °C. In vitro cell experiments showed that DON treated with the SIF containing 0.5% SMBS for 2 h completely attenuated the DON-induced cytotoxicity. When DON was treated with the SGF containing 0.5% encapsulated SMBS for 2 h and then the mixture was mixed with the SIF (1:1) and incubated for 2 h, it also completely attenuated the DON-induced cytotoxicity. Moreover, DON treated with the simulated fluid containing 0.5% encapsulated SMBS completely attenuated the gene expression inflammatory cytokines upregulated by DON and restored trans-epithelial electrical resistance (TEER) and tight junction and cytoskeleton. In summary, the encapsulation of SMBS was stable in SGF and allowed a progressive release of SMBS in the SIF. Moreover, the released SMBS in the SIF effectively attenuated the adverse effects induced by DON in the intestinal epithelial cells.

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Publisher's Expression of Concern: Lactobacillus bulgaricus Prevents Intestinal Epithelial Cell Injury Caused by Enterobacter sakazakii-Induced Nitric Oxide both In Vitro and in the Newborn Rat Model of Necrotizing Enterocolitis

Infection and Immunity ◽

10.1128/iai.00054-17 ◽

2017 ◽

Vol 85 (4) ◽

Author(s):

Keyword(s):

Nitric Oxide ◽

Epithelial Cell ◽

Rat Model ◽

Intestinal Epithelial Cell ◽

Cell Injury ◽

Lactobacillus Bulgaricus ◽

Intestinal Epithelial ◽

Enterobacter Sakazakii ◽

Newborn Rat

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Factors affecting the colonization of isolated rabbit intestinal epithelial cells by Vibrio cholerae 01 in vitro

Canadian Journal of Microbiology ◽

10.1139/m89-103 ◽

1989 ◽

Vol 35 (6) ◽

pp. 642-645 ◽

Cited By ~ 3

Author(s):

D. Sasmal ◽

B. Guhathakurta ◽

S. N. Sikdar ◽

A. Datta

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Vibrio Cholerae ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Growth Media ◽

Intestinal Epithelial ◽

Factors Affecting ◽

O Antigen

The adhesive capability of Vibrio cholerae 01 strains to isolated rabbit intestinal epithelial cells was maximally expressed when the bacteria were grown in synthetic broth and was enhanced by the presence of Ca2+ in the growth media. N-Acetyl-D-glucosamine could inhibit the adhesion of the bacteria to rabbit intestinal epithelial cells as could lipopolysaccharide O-antigen from Vibrio cholerae 01 and lectin from Triticum vulgaris. Since the lipopolysaccharide is known to contain N-acetyl-D-glucosamine and because the lectin from Triticum vulgaris shows specificity for this sugar, it is probable that N-acetyl-D-glucosamine is actively involved in the adhesion of Vibrio cholerae 01 to isolated rabbit intestinal epithelial cells.Key words: adhesion, Vibrio cholerae 01, rabbit intestinal epithelial cell.

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The Bacteroides fragilis Toxin Binds to a Specific Intestinal Epithelial Cell Receptor

Infection and Immunity ◽

10.1128/iai.00060-06 ◽

2006 ◽

Vol 74 (9) ◽

pp. 5382-5390 ◽

Cited By ~ 58

Author(s):

Shaoguang Wu ◽

Jai Shin ◽

Guangming Zhang ◽

Mitchell Cohen ◽

Augusto Franco ◽

...

Keyword(s):

Biological Activity ◽

Epithelial Cell ◽

Epithelial Cells ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Cellular Receptor ◽

Intestinal Epithelial ◽

Protease Activated Receptors ◽

E Cadherin

ABSTRACT The Bacteroides fragilis toxin (BFT) is the only known virulence factor of enterotoxigenic B. fragilis. BFT has previously been shown to act, at least in part, through cleavage of the intercellular adhesion protein E-cadherin. A specific cellular receptor for BFT has not been identified. The goal of this study was to determine if the initial interaction of BFT with intestinal epithelial cells was consistent with binding to a specific cellular receptor. Purified BFT was labeled with a fluorophore or iodide to assess specific cellular binding and the properties of BFT cellular binding. BFT binds specifically to intestinal epithelial cell lines in vitro in a polarized manner. However, specific binding occurs only at 37°C and requires BFT metalloprotease activity. The BFT receptor is predicted to be a membrane protein other than E-cadherin or a known protease-activated receptor (PAR1 to PAR4). BFT binding is resistant to acid washing, suggesting an irreversible interaction. Sugar or lipid residues do not appear to be involved in the mechanism of BFT cellular binding, but binding is sensitive to membrane cholesterol depletion. We conclude that intestinal epithelial cells in vitro possess a specific membrane BFT receptor that is distinct from E-cadherin. The data favor a model in which the metalloprotease domain of BFT processes its receptor protein, initiating cellular signal transduction that mediates the biological activity of BFT. However, activation of recognized protease-activated receptors does not mimic or block BFT biological activity or binding, suggesting that additional protease-activated receptors on intestinal epithelial cells remain to be identified.

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