scholarly journals RNAi-mediated silencing of Trichinella spiralis serpin-type serine protease inhibitors results in a reduction in larval infectivity

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Nana Yi ◽  
Pengcheng Yu ◽  
Lijia Wu ◽  
Zhaokun Liu ◽  
Jingzhe Guan ◽  
...  
Keyword(s):  
Survival Rate ◽  
Epithelial Cells ◽  
Serine Protease ◽  
Protease Inhibitors ◽  
Intestinal Epithelial Cells ◽  
Trichinella Spiralis ◽  
Intestinal Epithelial ◽  
Serine Protease Inhibitors

AbstractTrichinella spiralis serpin-type serine protease inhibitors (TsSPIs) are expressed in adult worms (AW), newborn larvae (NBL) and muscle larvae (ML) of T. spiralis, with the ML stage demonstrating the highest expression level. This study aims to determine TsSPI functions in larval viability and invasion of intestinal epithelial cells in vitro, as well as their development, survival, and fecundity in vivo via RNAi. TsSPI-specific siRNAs and dsRNA were transfected into ML by incubation. The silencing effect of TsSPI transcription and expression was determined using qPCR and western blot, respectively. After incubation in 60 ng/μL dsRNA–TsSPI for 3 days, larval TsSPI mRNA and protein expression levels were reduced by 68.7% and 68.4% (P < 0.05), respectively. dsRNA-mediated silencing of TsSPI significantly impacted larval invasion into intestinal epithelial cells in vitro but did not affect the survival rate of larvae. After challenge with dsRNA–TsSPI-treated ML, mice exhibited a 56.0% reduction in intestinal AW burden and 56.9% reduction in ML burden (P < 0.05), but NBL production of female AW remained the same (P > 0.05). Our results revealed that RNAi-mediated silencing of TsSPI expression in T. spiralis significantly reduced larval infectivity and survival in the host but had no effect on the survival rate and fecundity. Furthermore, TsSPIs have no effect on the growth and reproduction of parasites but may be directly involved in regulating the interaction of T. spiralis and the host. Therefore, TsSPIs are crucial in the process of T. spiralis larval invasion and parasite survival in the host.

Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

2003 ◽  
Vol 284 (2) ◽  
pp. G328-G339 ◽  
Author(s):  
P. Singh ◽  
X. Lu ◽  
S. Cobb ◽  
B. T. Miller ◽  
N. Tarasova ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Full Length ◽  
Intestinal Epithelial ◽  
Growth Effects ◽  
High Affinity ◽  
Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

7 LACTOBACILLUS REUTERI SUPPRESSES PRO-INFLAMMATORY DRIVEN REACTIVE OXYGEN SPECIES IN VITRO IN HUMAN INTESTINAL EPITHELIAL CELLS AND IN VIVO IN A TNBS COLITIS MOUSE MODEL

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S41-S41 ◽  
Author(s):  
Wenly Ruan ◽  
Melinda Engevik ◽  
Alexandra Chang-Graham ◽  
Joseph Hyser ◽  
James Versalovic
Keyword(s):  
Reactive Oxygen Species ◽  
Epithelial Cells ◽  
Lactobacillus Reuteri ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Oxygen Species ◽  
Intestinal Epithelial ◽  
Colitis Model

Abstract Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.

scholarly journals Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

2011 ◽  
Vol 49 (10) ◽  
pp. 1191-1200 ◽  
Author(s):  
Laura Carrillo ◽  
Ignacio Herrero ◽  
Inés Cambra ◽  
Rosa Sánchez-Monge ◽  
Isabel Diaz ◽  
...  
Keyword(s):  
Serine Protease ◽  
Protease Inhibitors ◽  
Serine Protease Inhibitors ◽  
Phytopathogenic Microorganisms

Flavonoids alter P-gp expression in intestinal epithelial cells in vitro and in vivo

2007 ◽  
Vol 51 (3) ◽  
pp. 293-300 ◽  
Author(s):  
Katrin Lohner ◽  
Kerstin Schnäbele ◽  
Hannelore Daniel ◽  
Doris Oesterle ◽  
Gerhard Rechkemmer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial

Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells

2002 ◽  
Vol 48 (5) ◽  
pp. 449-457 ◽  
Author(s):  
Gabriela Zárate ◽  
Vilma Morata De Ambrosini ◽  
Adriana Perez Chaia ◽  
Silvia González
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Divalent Cations ◽  
Metabolic Effects ◽  
Bacterial Cells ◽  
Intestinal Epithelial ◽  
Propionibacterium Acidipropionici ◽  
Sodium Metaperiodate

Adhesion to the intestinal mucosa is generally considered an important property of probiotic microorganisms and has been related to many of their health benefits. This study investigated some factors that could affect or be involved in the adherence of Propionibacterium acidipropionici CRL 1198, a dairy strain with suggested probiotic effects and high adherence in vitro and in vivo to intestinal epithelial cells. In vitro adhesion of propionibacteria was decreased by gastric digestion but not affected by bile and pancreatic enzymes. Adherence was also decreased by pretreatment of bacterial cells with protease, sodium metaperiodate, and trichloroacetic acid, revealing that different features of the cell surface, like protein factors, carbohydrates, and teichoic acids, are involved in the process. Adherence to intestinal epithelial cells was enhanced by calcium and was dependent on other divalent cations. Adhesion to intestinal mucus was also demonstrated. The results should explain the metabolic effects in the host previously obtained with this strain and support the potential of Propionibacterium for development of new probiotics.Key words: propionibacteria, adhesion, probiotics.

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