The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors.

• MeSH
• algoritmy MeSH
• beta-katenin chemie   metabolismus   MeSH
• buněčná diferenciace MeSH
• chromatin metabolismus   MeSH
• fenotyp MeSH
• genetická transkripce * MeSH
• homeostáza MeSH
• hyperplazie MeSH
• JNK mitogenem aktivované proteinkinasy metabolismus   MeSH
• kmenové buňky metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace genetika   MeSH
• mutantní proteiny metabolismus   MeSH
• myši MeSH
• organoidy metabolismus   MeSH
• proliferace buněk MeSH
• restrukturace chromatinu MeSH
• sekvence nukleotidů MeSH
• signální transdukce MeSH
• střevní sliznice cytologie   MeSH
• transkripční faktory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

• MeSH
• aurora kinasa B metabolismus   MeSH
• azoxymethan toxicita   MeSH
• chemorezistence genetika   MeSH
• chromozomální nestabilita * MeSH
• cytogenetické vyšetření MeSH
• dextrany toxicita   MeSH
• experimentální nádory chemicky indukované   genetika   patologie   MeSH
• genový knockdown MeSH
• karcinogeneze genetika   MeSH
• kolon cytologie   patologie   MeSH
• kolorektální nádory chemicky indukované   genetika   patologie   MeSH
• lidé MeSH
• myši transgenní MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• organoidy MeSH
• primární buněčná kultura MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• protokoly antitumorózní kombinované chemoterapie farmakologie   terapeutické užití   MeSH
• protoonkogenní proteiny c-myc metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• RNA dlouhá nekódující genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• střevní sliznice cytologie   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The ability to adhere via colonization factors to specific receptors located on the intestinal mucosa is a key virulence factor in enterotoxigenic Escherichia coli (ETEC) pathogenesis. Here, the potential glycosphingolipid receptors of the novel human ETEC colonization factor CS30 were examined by binding of CS30-expressing bacteria to glycosphingolipids on thin-layer chromatograms. We thereby found a highly specific binding of CS30-expressing bacteria to a fast-migrating acid glycosphingolipid of human and porcine small intestine, while no binding was obtained with a mutant ETEC strain unable to express CS30 fimbriae. The CS30 binding glycosphingolipid from human small intestine was isolated and characterized by mass spectrometry as sulfatide (SO3-3Galβ1Cer). Comparative binding studies using sulfatides with different ceramide compositions gave a preferential binding of CS30 to sulfatide with d18:1-h24:0 ceramide. This ceramide species of sulfatide was also isolated from human small intestine and characterized by mass spectrometry and antibody binding. These studies implicate sulfatide as candidate receptor for mediating attachment of CS30-fimbriated ETEC to human and porcine small intestinal cells. Our findings may be a basis for designing receptor saccharide analogues for inhibition of the intestinal adhesion of CS30-expressing E. coli.

• MeSH
• bakteriální adheze * MeSH
• ceramidy analýza   MeSH
• enterotoxigenní Escherichia coli metabolismus   MeSH
• faktory virulence metabolismus   MeSH
• glykosfingolipidy metabolismus   MeSH
• lidé MeSH
• prasata MeSH
• proteiny fimbrií genetika   metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• střevní sliznice cytologie   mikrobiologie   MeSH
• sulfoglykosfingolipidy metabolismus   MeSH
• tenké střevo cytologie   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Trans-resveratrol, a well-known plant phenolic compound, has been intensively investigated due to its association with the so-called French paradox. However, despite its high pharmacological potential, trans-resveratrol has shown relatively low bioavailability. Trans-resveratrol is intensively metabolized in the intestine and liver, yielding metabolites that may be responsible for its high bioactivity. The aim of this study was to investigate and compare the metabolism of trans-resveratrol (tRes), cis-resveratrol (cRes) and dihydroresveratrol (dhRes) in an in vitro epithelial model using Caco-2 cell lines. Obtained metabolites of tRes, cRes and dhRes were analyzed by LC/MS Q-TOF, and significant differences in the metabolism of each compound were observed. The majority of tRes was transported unchanged through the Caco-2 cells, while cRes was mostly metabolized. The main metabolite of both cis- and trans-resveratrol observed as a result of colon microbial metabolism, dhRes, was metabolized almost completely, with only traces of the unchanged molecule being found. A sulphate conjugate was identified as the main metabolite of tRes in our model, while a glucuronide conjugate was the major metabolite of cRes and dhRes. Since metabolism of simple phenolics and polyphenols plays a crucial role in their bioavailability, detailed knowledge of their transformation is of high scientific value.

• MeSH
• biologická dostupnost MeSH
• Caco-2 buňky MeSH
• lidé MeSH
• permeabilita MeSH
• resveratrol chemie   farmakokinetika   MeSH
• stereoizomerie MeSH
• stilbeny chemie   farmakokinetika   MeSH
• střevní sliznice cytologie   metabolismus   mikrobiologie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Dietary and microbial indoles can act as ligands and activators of pregnane X receptor (PXR), with implications in human intestinal health. In the current study, we examined the effects of simple mono-methylated indoles (MMIs) on the activity and function of PXR, using a series of human hepatic and intestinal cell models. Indoles 1-MMI and 2-MMI strongly induced CYP3A4 and MDR1 mRNAs in human intestinal adenocarcinoma cells LS180, but not in primary human hepatocytes. The levels of CYP3A4 mRNA were increased by 1-MMI and 2-MMI in wild type, but not in PXR-knock-out human hepatic progenitor HepaRG cells, implying the involvement of PXR in CYP3A4 induction by MMIs. Utilizing reporter gene assay, we observed dose-dependent activation of PXR by all MMIs, and their efficacies and potencies were comparable. Tested MMIs also displayed moderate antagonist effects on PXR, revealing about partial agonist effects of these compounds. As demonstrated using the Chromatin immunoprecipitation assay (ChIP),1-MMI increased PXR occupancy of the CYP3A4 promoter. Time-Resolved Fluorescence Resonance Energy Transfer revealed that MMIs are weak ligands of human PXR. Collectively, we show that MMIs are ligands and partial agonists of human PXR, which induce PXR-regulated genes in human intestinal cells.

• MeSH
• cytochrom P-450 CYP3A genetika   fyziologie   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• indoly metabolismus   farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• P-glykoproteiny genetika   MeSH
• pregnanový X receptor účinky léků   genetika   metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• signální transdukce účinky léků   MeSH
• střevní sliznice cytologie   účinky léků   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The objective of this work was to compare the effect of selected feed mixtures on the duodenal morphology. One-hundred-four rats of the Wistar strain were divided to thirteen groups per eight rats. The experiment started in 35-day-old rats after birth and lasted for 32 days. The groups (A-M) were fed by commercial diet, 85 % wheat and 15 % oat diet, 85 % wheat and 15 % triticale, 85 % wheat and 15 % barley, 85 % wheat and 15 % amaranth, 85 % wheat and 15 % lantern, 85 % wheat and 15 % buckwheat, 100 % wheat, 100 % white lupine, 100 % flock peas - variety Garden, 100 % native peas - variety Garden, 100 % native peas - variety Zekon or 100 % extruded peas - variety Zekon diet, respectively. Samples from the duodenum were taken. The height of the villi and the depth of the crypts were measured. The tallest villi were measured in group F (474.33+/-114.36 microm) and the shortest villi were observed in group B (294.08+/-88.52 microm). The deepest crypts were measured in group K (166.41+/-35.69 microm) and the shallowest crypts were observed in group E (77.85+/-17.61 microm). The work documents that gluten-free and classical cereals combination can be a better choice for people who want to limit the gluten content of the diet.

• MeSH
• bezlepková dieta metody   MeSH
• duodenum cytologie   patologie   MeSH
• hrách setý * škodlivé účinky   MeSH
• jedlá semena * škodlivé účinky   MeSH
• krmivo pro zvířata * škodlivé účinky   MeSH
• krysa rodu rattus MeSH
• potkani Wistar MeSH
• střevní sliznice cytologie   patologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Proinflammatory cytokines play important roles in the pathogenesis of diseases caused by enterohemorrhagic Escherichia coli (EHEC) O157, but the spectrum of bacterial components involved in the proinflammatory responses is not fully understood. Here, we investigated the abilities of outer membrane vesicles (OMVs), nanoparticles released by EHEC O157 during growth, to induce production of proinflammatory cytokines in human intestinal epithelial cells. OMVs from both EHEC O157:H7 and sorbitol-fermenting (SF) EHEC O157:H- induced production of interleukin-8 (IL-8) in Caco-2, HCT-8, and HT-29 intestinal epithelial cell lines. H7 flagellin was the key IL-8-inducing component of EHEC O157:H7 OMVs, whereas cytolethal distending toxin V and O157 lipopolysaccharide (LPS) largely contributed to IL-8 production elicited by flagellin-lacking OMVs from SF EHEC O157:H-. The H7 flagellin-mediated signaling via Toll-like receptor (TLR) 5, and O157 LPS-mediated signaling via TLR4/MD-2 complex, which were followed by activation of the nuclear factor NF-κB were major pathways underlying IL-8 production induced by EHEC O157 OMVs. The proinflammatory and immunomodulatory capacities of EHEC O157 OMVs have pathogenetic implications and support the OMVs as suitable vaccine candidates.

• MeSH
• buněčná membrána metabolismus   MeSH
• buňky HT-29 MeSH
• Caco-2 buňky MeSH
• epitelové buňky metabolismus   MeSH
• Escherichia coli O157 patogenita   MeSH
• faktory virulence metabolismus   MeSH
• flagelin metabolismus   MeSH
• infekce vyvolané Escherichia coli mikrobiologie   patologie   MeSH
• interleukin-8 biosyntéza   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• NF-kappa B metabolismus   MeSH
• proteiny vnější bakteriální membrány metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• signální transdukce MeSH
• střevní sliznice cytologie   mikrobiologie   patologie   MeSH
• toll-like receptor 4 metabolismus   MeSH
• toll-like receptor 5 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

• MeSH
• beta-katenin biosyntéza   MeSH
• buněčná diferenciace účinky léků   MeSH
• cytokiny biosyntéza   MeSH
• epitelové buňky účinky léků   MeSH
• interleukin-18 biosyntéza   MeSH
• kaspasa 1 biosyntéza   MeSH
• krysa rodu rattus MeSH
• Lacticaseibacillus rhamnosus * MeSH
• Lactobacillus casei * MeSH
• messenger RNA biosyntéza   MeSH
• mikrofilamentové proteiny biosyntéza   MeSH
• probiotika farmakologie   MeSH
• regulace genové exprese účinky léků   MeSH
• střevní sliznice cytologie   účinky léků   MeSH
• subcelulární frakce metabolismus   MeSH
• toll-like receptory biosyntéza   účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The Wnt pathway plays a crucial role in self-renewal and differentiation of cells in the adult gut. In the present study, we revealed the functional consequences of inhibition of canonical Wnt signaling in the intestinal epithelium. The study was based on generation of a novel transgenic mouse strain enabling inducible expression of an N-terminally truncated variant of nuclear Wnt effector T cell factor 4 (TCF4). The TCF4 variant acting as a dominant negative (dn) version of wild-type (wt) TCF4 protein decreased transcription of β-catenin-TCF4-responsive genes. Interestingly, suppression of Wnt/β-catenin signaling affected asymmetric division of intestinal stem cells (ISCs) rather than proliferation. ISCs expressing the transgene underwent several rounds of division but lost their clonogenic potential and migrated out of the crypt. Expression profiling of crypt cells revealed that besides ISC-specific markers, the dnTCF4 production downregulated expression levels of epithelial genes produced in other crypt cells including markers of Paneth cells. Additionally, in Apc conditional knockout mice, dnTCF activation efficiently suppressed growth of Apc-deficient tumors. In summary, the generated mouse strain represents a convenient tool to study cell-autonomous inhibition of β-catenin-Tcf-mediated transcription.

• MeSH
• beta-katenin metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné dělení MeSH
• genetická transkripce MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• myši transgenní MeSH
• myši MeSH
• proliferace buněk MeSH
• signální dráha Wnt * MeSH
• střevní sliznice cytologie   metabolismus   MeSH
• tenké střevo cytologie   metabolismus   MeSH
• transkripční faktory BHLH-Zip chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Crohn's disease (CD) is a disabling chronic enteropathy sustained by a harmful T-cell response toward antigens of the gut microbiota in genetically susceptible subjects. Growing evidence highlights the safety and possible efficacy of mesenchymal stem cells (MSCs) as a new therapeutic tool for this condition. Therefore, we aimed to investigate the effects of bone marrow-derived MSCs on pathogenic T cells with a view to clinical application. METHODS: T-cell lines from both inflamed and non-inflamed colonic mucosal specimens of CD patients and from healthy mucosa of control subjects were grown with the antigen muramyl-dipeptide in the absence or presence of donors' MSCs. The MSC effects were evaluated in terms of T-cell viability, apoptotic rate, proliferative response, immunophenotype, and cytokine profile. The role of the indoleamine 2,3-dioxygenase (IDO) was established by adding a specific inhibitor, the 1-methyl-DL-tryptophan, and by using MSCs transfected with the small interfering RNA (siRNA) targeting IDO. The relevance of cell-cell contact was evaluated by applying transwell membranes. RESULTS: A significant reduction in both cell viability and proliferative response to muramyl-dipeptide, with simultaneous increase in the apoptotic rate, was found in T cells from both inflamed and non-inflamed CD mucosa when co-cultured with MSCs and was reverted by inhibiting IDO activity and expression. A reduction of the activated CD4(+)CD25(+) subset and increase of the CD3(+)CD69(+) population were also observed when T-cell lines from CD mucosa were co-cultured with MSCs. In parallel, an inhibitory effect was evident on the expression of the pro-inflammatory cytokines tumor necrosis factor-α, interferon-γ, interleukin-17A and -21, whereas that of the transforming growth factor-β and interleukin-6 were increased, and production of the tolerogenic molecule soluble HLA-G was high. These latter effects were almost completely eliminated by blocking the IDO, whose activity was upregulated in MSCs co-cultured with CD T cells. The use of a semipermeable membrane partially inhibited the MSC immunosuppressive effects. Finally, hardly any effects of MSCs were observed when T cells obtained from control subjects were used. CONCLUSION: MSCs exert potent immunomodulant effects on antigen-specific T cells in CD through a complex paracrine and cell-cell contact-mediated action, which may be exploited for widespread therapeutic use.

• MeSH
• acetylmuramyl-alanyl-isoglutamin farmakologie   MeSH
• antigeny povrchové metabolismus   MeSH
• apoptóza účinky léků   MeSH
• buňky kostní dřeně cytologie   MeSH
• časosběrné zobrazování MeSH
• Crohnova nemoc patologie   MeSH
• cytokiny metabolismus   MeSH
• dospělí MeSH
• HLA-G antigeny metabolismus   MeSH
• imunofenotypizace MeSH
• indolamin-2,3,-dioxygenasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• kokultivační techniky MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• proliferace buněk účinky léků   MeSH
• RNA interference MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• střevní sliznice cytologie   MeSH
• T-lymfocyty cytologie   účinky léků   imunologie   MeSH
• tryptofan analogy a deriváty   farmakologie   MeSH
• viabilita buněk MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH