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1.
Toxicol Lett ; 324: 104-110, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32092453

RESUMO

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.


Assuntos
Hepatócitos/efeitos dos fármacos , Indóis/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Receptor de Pregnano X/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/fisiologia , Hepatócitos/metabolismo , Humanos , Indóis/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos
2.
J Agric Food Chem ; 68(1): 160-167, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31825618

RESUMO

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of intestinal mucosa and submucosa, characterized by the disruption of the intestinal epithelial barrier, increased production of inflammatory mediators, and excessive tissue injury. Intestinal epithelial cells, as well as microvascular endothelial cells, play important roles in IBD. To study the potential effects of kaempferol in IBD progress, we established a novel epithelial-endothelial cells coculture model to investigate the intestinal inflammation and barrier function. Data demonstrated an obvious increased transepithelial electrical resistance (TEER) (1222 ± 60.40 Ω cm2 vs 1371 ± 38.77 Ω cm2), decreased flux of FITC (180.8 ± 20.06 µg/mL vs 136.7 ± 14.78 µg/mL), and up-regulated occludin and claudin-2 expression in Caco-2 that was specifically cocultured with endothelial cells. Meanwhile, 80 µM kaempferol alleviated the drop of TEER, the increase of FITC flux, and the overexpression of interleukin-8 (IL-8) induced by 1 µg/mL lipopolysaccharide (LPS). Additionally, kaempferol also ameliorated the LPS-induced decrease of protein expression of zonula occludens-1 (ZO-1), occludin, and claudin-2, together with the inhibited protein expressions of the phosphorylation level of NF-κB and I-κB induced by LPS. Our results suggest that kaempferol alleviates the IL-8 secretion and barrier dysfunction of the Caco-2 monolayer in the LPS-induced epithelial-endothelial coculture model via inhibiting the NF-κB signaling pathway activation.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/citologia , Quempferóis/farmacologia , Lipopolissacarídeos/efeitos adversos , Células CACO-2 , Claudina-2/genética , Claudina-2/metabolismo , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/imunologia , Microvilosidades/efeitos dos fármacos , Microvilosidades/genética , Microvilosidades/metabolismo , Ocludina/genética , Ocludina/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
3.
Nihon Saikingaku Zasshi ; 74(3): 167-175, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31787706

RESUMO

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum and related species cause botulism, a neuroparalytic disease associated with a high mortality. BoNTs are always produced as large protein complexes (progenitor toxin complexes, PTCs) through association with non-toxic components (NAPs) including hemagglutinin (HA) and non-toxic non-hemagglutinin (NTNHA). Food-borne botulism is caused by the ingestion of PTCs. PTCs in the gastrointestinal tract cross the intestinal epithelial barrier, enter the blood stream, and reach the nerve endings, where BoNTs cleave the SNAREs required for vesicle fusion. Consequently, BoNTs inhibit neurotransmitter release and cause paralysis. To cause food-borne botulism, BoNTs must traverse the intestinal epithelial barrier. However, the mechanism used to cross this barrier remains unclear. Using an in vitro epithelial barrier system, we previously showed that the interaction of HA with E-cadherin results in disruption of tight junctions. Furthermore, we previously reported that microfold (M) cells in the follicle-associated epithelium (FAE) of mouse Peyer's patches (PPs) are major sites where type A1 BoNT breaches the intestinal epithelial barrier. Here, I would like to demonstrate an ingenious invasion mechanism of the BoNT complex.


Assuntos
Toxinas Botulínicas/metabolismo , Células Epiteliais/metabolismo , Absorção Intestinal , Mucosa Intestinal/citologia , Complexos Multiproteicos/metabolismo , Neurotoxinas/metabolismo , Animais , Caderinas , Células Cultivadas , Cães , Hemaglutininas , Humanos , Camundongos , Terminações Nervosas/metabolismo , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Nódulos Linfáticos Agregados/metabolismo , Proteínas SNARE/metabolismo
4.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(10): 892-896, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31814565

RESUMO

Objective To investigate the effect of miR-129-5p on alcohol-induced intestinal epithelial permeability. Methods Real-time quantitative PCR was used to detect the expression level of miR-129-5p in sigmoid colon biopsies and alcohol-induced differentiated Caco-2 cells from patients with alcoholic liver disease and healthy people. Then, the differentiated Caco-2 cells were divided into 4 groups: the control group, the alcohol group (treatment with 50 mmol/L alcohol for 60 minutes), alcohol combined with miR-129-5p inhibitor group (transfection with miR-129-5p inhibitor before alcohol treatment), and alcohol combined with miR-129-5p inhibitor control group (transfection with miR-129-5p inhibitor control before alcohol treatment). After the alcohol treatment, the transepithelial electrical resistance (TER) was analyzed by a resistance meter, and the protein levels relevant to tight junction (occludin and ZO-1) were determined by Western blotting. Results The expression level of miR-129-5p was elevated in the biopsies of the patients with alcoholic liver disease compared with that in the healthy controls. Alcohol treatment increased miR-129-5p expression in the differentiated Caco-2 cells. Compared with the control group, TER and the protein levels of occludin and ZO-1 decreased in the alcohol group. Compared with the alcohol group, TER and the protein levels of occludin and ZO-1 were enhanced in the alcohol combined with miR-129-5p group; however, these changes were not altered in the alcohol combined with miR-129-5p inhibitor control group. Conclusion The miR-129-5p expression is elevated in sigmoid colon biopsies and alcohol-induced differentiated Caco-2 cells from patients with alcoholic liver disease. Inhibition of miR-129-5p can suppress alcohol-induced intestinal epithelial permeability.


Assuntos
Células Epiteliais/efeitos dos fármacos , Etanol/farmacologia , Mucosa Intestinal/citologia , MicroRNAs/genética , Células CACO-2 , Regulação para Baixo , Células Epiteliais/citologia , Humanos , Ocludina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
5.
PLoS Genet ; 15(12): e1008553, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31841513

RESUMO

Many tissues rely on resident stem cell population to maintain homeostasis. The balance between cell proliferation and differentiation is critical to permit tissue regeneration and prevent dysplasia, particularly following tissue damage. Thus, understanding the cellular processes and genetic programs that coordinate these processes is essential. Here, we report that the conserved transcription factor zfh2 is specifically expressed in Drosophila adult intestinal stem cell and progenitors and is a critical regulator of cell differentiation in this lineage. We show that zfh2 expression is required and sufficient to drive the activation of enteroblasts, the non-proliferative progenitors of absorptive cells. This transition is characterized by the transient formation of thin membrane protrusions, morphological changes characteristic of migratory cells and compensatory stem cell proliferation. We found that zfh2 acts in parallel to insulin signaling and upstream of the TOR growth-promoting pathway during early differentiation. Finally, maintaining zfh2 expression in late enteroblasts blocks terminal differentiation and leads to the formation of highly dysplastic lesions, defining a new late cell differentiation transition. Together, our study greatly improves our understanding of the cascade of cellular changes and regulatory steps that control differentiation in the adult fly midgut and identifies zfh2 as a major player in these processes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Mucosa Intestinal/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Proliferação de Células , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica , Insulina/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo
6.
Vet Res ; 50(1): 110, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856906

RESUMO

Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular diffusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifically infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) significantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our findings demonstrate that integrity of the intestinal epithelium is crucial in the host's innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface.


Assuntos
Enterócitos/virologia , Células Epiteliais/virologia , Mucosa Intestinal/citologia , Rotavirus/classificação , Rotavirus/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/veterinária , Ácido Egtázico/farmacologia , Enterócitos/efeitos dos fármacos , Miofibroblastos/fisiologia , Suínos , Internalização do Vírus , Replicação Viral
7.
PLoS Pathog ; 15(10): e1008057, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31671153

RESUMO

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.


Assuntos
Infecções por Astroviridae/imunologia , Infecções por Astroviridae/veterinária , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Mamastrovirus/fisiologia , Tropismo Viral/genética , Animais , Células CACO-2 , Linhagem Celular , Enterócitos/virologia , Gastroenterite/virologia , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/virologia , Intestino Delgado/citologia , Intestino Delgado/virologia , Mamastrovirus/genética , Mamastrovirus/imunologia , Células Vero , Tropismo Viral/imunologia
8.
World J Gastroenterol ; 25(38): 5800-5813, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31636473

RESUMO

BACKGROUND: Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase that is involved in various diseases, including cancers, metabolic diseases, and inflammation-associated diseases. However, the role of SIRT1 in ulcerative colitis (UC) is still confusing. AIM: To investigate the role of SIRT1 in intestinal epithelial cells (IECs) in UC and further explore the underlying mechanisms. METHODS: We developed a coculture model using macrophages and Caco-2 cells. After treatment with the SIRT1 activator SRT1720 or inhibitor nicotinamide (NAM), the expression of occludin and zona occludens 1 (ZO-1) was assessed by Western blot analysis. Annexin V-APC/7-AAD assays were performed to evaluate Caco-2 apoptosis. Dextran sodium sulfate (DSS)-induced colitis mice were exposed to SRT1720 or NAM for 7 d. Transferase-mediated dUTP nick-end labeling (TUNEL) assays were conducted to assess apoptosis in colon tissues. The expression levels of glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, caspase-9, and caspase-3 in Caco-2 cells and the colon tissues of treated mice were examined by quantitative real-time PCR and Western blot. RESULTS: SRT1720 treatment increased the protein levels of occludin and ZO-1 and inhibited Caco-2 apoptosis, whereas NAM administration caused the opposite effects. DSS-induced colitis mice treated with SRT1720 had a lower disease activity index (P < 0.01), histological score (P < 0.001), inflammatory cytokine levels (P < 0.01), and apoptotic cell rate (P < 0.01), while exposure to NAM caused the opposite effects. Moreover, SIRT1 activation reduced the expression levels of GRP78, CHOP, cleaved caspase-12, cleaved caspase-9, and cleaved caspase-3 in Caco-2 cells and the colon tissues of treated mice. CONCLUSION: SIRT1 activation reduces apoptosis of IECs via the suppression of endoplasmic reticulum stress-mediated apoptosis-associated molecules CHOP and caspase-12. SIRT1 activation may be a potential therapeutic strategy for UC.


Assuntos
Apoptose , Colite Ulcerativa/patologia , Estresse do Retículo Endoplasmático , Mucosa Intestinal/patologia , Sirtuína 1/metabolismo , Animais , Células CACO-2 , Caspase 12/metabolismo , Técnicas de Cocultura , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Humanos , Mucosa Intestinal/citologia , Macrófagos , Camundongos , Niacinamida/administração & dosagem , Sirtuína 1/antagonistas & inibidores , Fator de Transcrição CHOP/metabolismo
9.
Vet Microbiol ; 237: 108400, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31585640

RESUMO

The entry mechanism of porcine epidemic diarrhea virus (PEDV) remains unclear, especially the virus receptor. Our previous study revealed a potential correlation between integrin αvß3 and PEDV infection. In the current study, the effect of overexpression, silencing, antibody inhibition, and co-expression with porcine aminopeptidase N (pAPN) of integrin αvß3 on PEDV infection was investigated and analyzed in African green monkey Vero E6 cells and porcine intestinal epithelial cells (IECs) using the classical strain CV777 and variant strain HM2017 of PEDV. Integrin αvß3 significantly enhanced the replication of the classical and variant strains of PEDV in Vero E6 cells and IECs. The integrin αv and ß3 subunits were both involved in the enhancement of PEDV infection, the Arg-Gly-Asp peptides targeting integrin αvß3 significantly inhibited replication of PEDV in Vero E6 cells, and co-expression of integrin αvß3 with pAPN significantly enhanced replication of PEDV in Vero E6 and BHK-21 cells. These results demonstrate that integrin αvß3 enhances PEDV replication in Vero E6 cells and IECs. These data provide novel insights into the entry mechanism of PEDV.


Assuntos
Células Epiteliais/virologia , Integrina alfaVbeta3/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Cultura de Vírus/veterinária , Replicação Viral/fisiologia , Animais , Regulação da Expressão Gênica , Inativação Gênica , Mucosa Intestinal/citologia , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Células Vero
10.
World J Gastroenterol ; 25(36): 5469-5482, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31576093

RESUMO

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most common functional gas-troenterological diseases characterized by abnormal visceral sensitivity and low-grade inflammation. The role of Clostridium butyricum (C. butyricum) in reducing intestinal low-grade inflammation via immune pathways has been well defined. However, the detailed mechanisms of the effects of C. butyricum on intestinal mucosal immunity, especially on immune cells of the lamina propria, remain unclear. Dendritic cells (DCs), which are important immune cells, secrete proinflammatory cytokines (IL-1ß, IL-6, and others) and express T cell immuno-globulin and mucin domain-3 (TIM3), promoting proliferation and activation of DCs, and mediating Th1 and Th17 inflammatory responses. AIM: To investigate the role of DCs in the development of IBS in a rat model and to understand the regulation of DCs after C. butyricum intervention. METHODS: An IBS animal model was established using C57BL/6 mice, and C. butyricum was continuously administered via the intragastric route to simulate different intestinal immune states. Intestinal visceral hypersensitivity and histopathology were assessed using the abdominal withdrawal reflex (AWR) test and hematoxylin & eosin (H&E) staining, respectively. The expression of proinflammatory cytokines (IL-1ß and IL-6) and TIM3 was analyzed by Western blot analysis and real-time PCR. Flow cytometry was applied to analyze the quantity, function, and membrane molecule TIM3 of the lamina propria dendritic cells (LPDCs). The regulatory effect of C. butyricum was verified in bone marrow-derived dendritic cells by in vitro experiments. RESULTS: The secretion of proinflammatory cytokines (IL-1ß and IL-6) in mice with IBS was significantly increased compared with that of the control group, which suggested that the intestinal mucosa in mice with IBS was in a low-grade inflammatory state. The expression of CD11C+CD80+ and CD11c+TIM3+ in intestinal LPDCs in mice with IBS increased significantly. Meanwhile, the cytokines (IL-1ß and IL-6) were significantly reduced after the intervention with probiotic C. butyricum. The amount and function of LPDCs and the TIM3 on the surface of the LPDCs were decreased with the alleviation of the intestinal inflammatory response. CONCLUSION: The results suggest that C. butyricum regulates the amount and functional status of LPDCs in the intestinal mucosa of mice with IBS, and therefore modulates the local immune response in the intestine.


Assuntos
Clostridium butyricum/imunologia , Células Dendríticas/imunologia , Mucosa Intestinal/imunologia , Síndrome do Intestino Irritável/terapia , Probióticos/administração & dosagem , Animais , Modelos Animais de Doenças , Humanos , Imunidade nas Mucosas , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Síndrome do Intestino Irritável/induzido quimicamente , Síndrome do Intestino Irritável/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Resultado do Tratamento , Ácido Trinitrobenzenossulfônico/toxicidade
11.
Fitoterapia ; 139: 104367, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31629045

RESUMO

Ca2+-activated Cl- channels (CaCCs) wildly exist in many tissues which play an important role in ion transport and excitation conduction, especially fluid secretion and smooth muscle contraction in epithelial tissues. TMEM16A as a classic CaCC expresses in the intestine, and has become a potential target of intestinal physiological and pathological researches and therapeutic drug screening. In this study, we identified trans-δ-viniferin (TVN), a resveratrol dimmer, could inhibit TMEM16A activity in TMEM16A expressed FRT cells with IC50 of 19.7 µM, it also prevented Ca2+-activated Cl- current in HT-29 cells with IC50 of 4.65 µM and in colonic mucosa. In the mechanism studies, TVN showed no significant inhibition on CFTR and basal Na+/K+-ATPase in both intestinal epithelial cells and colonic tissues, except for inhibition of calcium concentration and Ca2+-activated K+ channel to some degree. In anti-diarrheal studies, TVN could effectively prevent diarrhea caused by rotavirus infection and reduce the pellet number in IBS-D mice. These physiological effects are at least partially attributed to the inhibitory effect of TVN on CaCC-mediated intestinal fluid secretion and the reduction of smooth muscle contraction force by inhibiting TMEM16A. Collectively, the present study identified a new pharmacological target of TVN which provided the theoretical basis for the application of TVN in the treatment of rotavirus-infected diarrhea and IBS-D.


Assuntos
Benzofuranos/farmacologia , Canais de Cloreto/antagonistas & inibidores , Diarreia/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Resorcinóis/farmacologia , Estilbenos/farmacologia , Animais , Cálcio/análise , Diarreia/virologia , Motilidade Gastrointestinal/efeitos dos fármacos , Células HT29 , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucosa Intestinal/citologia , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ratos , Rotavirus
12.
Nature ; 574(7779): 532-537, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645730

RESUMO

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.


Assuntos
Colo/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mutação , Sintomas Prodrômicos , Reto/citologia , Adenoma/genética , Adenoma/patologia , Idoso , Proteína Axina/genética , Carcinoma/genética , Carcinoma/patologia , Transformação Celular Neoplásica , Células Clonais/citologia , Células Clonais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Células-Tronco/citologia , Células-Tronco/metabolismo
13.
Nucleic Acids Res ; 47(19): 10059-10071, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501873

RESUMO

Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode Caenorhabditis elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells. However, the lineal origins of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. Variability is masked in wild-type animals, which show extensive silencing by neuronal dsRNA. As a result, cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against inferring uniformity of mechanism based on uniformity of outcome. We speculate that functional mosaicism could contribute to escape from targeted therapies and could allow developmental systems to drift over evolutionary time.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Inativação Gênica , RNA Replicase/genética , RNA de Cadeia Dupla/genética , Animais , Animais Geneticamente Modificados/genética , Caenorhabditis elegans/genética , Linhagem da Célula/genética , Células Germinativas , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mosaicismo , Neurônios/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
14.
Gastroenterology ; 157(6): 1584-1598, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31513797

RESUMO

BACKGROUND & AIMS: T-regulatory (Treg) cells suppress the immune response to maintain homeostasis. There are 2 main subsets of Treg cells: FOXP3 (forkhead box protein 3)-positive Treg cells, which do not produce high levels of effector cytokines, and type 1 Treg (Tr1) cells, which are FOXP3-negative and secrete interleukin (IL) 10. IL10 is an anti-inflammatory cytokine, so Tr1 cells might be used in the treatment of inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions. METHODS: We obtained blood and colon biopsy samples from patients with Crohn's disease or ulcerative colitis or healthy individuals (controls). CD4+ T cells were isolated from blood samples and stimulated with anti-CD3 and anti-CD28 beads, and Tr1 cells were purified by using an IL10 cytokine-capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4+CD25highCD127low cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3 and anti-CD28 beads, and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colon organoid cultures were established, cultured with supernatants from Tr1 or FOXP3-positive cells, and analyzed by immunofluorescence and flow cytometry. T84 cells (human colon adenocarcinoma epithelial cells) were incubated with supernatants from Tr1 or FOXP3-positive cells, and transepithelial electrical resistance was measured to determine epithelial cell barrier function. RESULTS: Phenotypes of Tr1 cells isolated from control individuals vs patients with Crohn's disease or ulcerative colitis did not differ significantly after expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1B and tumor necrosis factor from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy individuals and patients with Crohn's disease or ulcerative colitis secreted IL22, which promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures. CONCLUSIONS: Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (inmate immune response). They also secrete IL22 to promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders.


Assuntos
Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Interleucina-10/metabolismo , Mucosa Intestinal/patologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Biópsia , Comunicação Celular/imunologia , Proliferação de Células , Células Cultivadas , Colite Ulcerativa/sangue , Colite Ulcerativa/terapia , Colo/citologia , Colo/imunologia , Colo/patologia , Doença de Crohn/sangue , Doença de Crohn/terapia , Feminino , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Voluntários Saudáveis , Humanos , Interleucina-10/imunologia , Interleucinas/imunologia , Interleucinas/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplante
15.
J Biochem Mol Toxicol ; 33(11): e22397, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31557363

RESUMO

Fumonisins (Fums) are mycotoxins widely distributed in crops and feed, and ingestion of Fums-contaminated crops is harmful to animal health. The purpose of this study is to explore the effect of Fum B1 (FB1 ) on barrier functions of porcine intestinal epithelial cells, IPEC-J2, to clarify the intestinal toxicity of Fums in pigs. The results showed that the persistent treatment of FB1 significantly decreased the viability of IPEC-J2. Moreover, the expressions of Claudin 1, Occludin, Zonula Occluden-1 (ZO-1) on the messenger RNA (mRNA), and protein levels and MUC1 on the mRNA level were significantly inhibited after FB1 treatment, while the mRNA relative expression level of MUC2 was clearly increased. FB1 also enhanced the monolayer cell permeability of IPEC-J2. Importantly, FB1 promoted the expression of phosphorylated extracellular regulated protein kinase (p-ERK1/2 ). These data suggest that long-term treatment of FB1 can suppress IPEC-J2 proliferation, damage tight junctions of IPEC-J2, and regulate expression of mucins to induce the damage of barrier functions of porcine intestinal epithelial cells, which may be associated with the ERK1/2 phosphorylation pathway.


Assuntos
Células Epiteliais/metabolismo , Fumonisinas/farmacologia , Mucosa Intestinal/citologia , Micotoxinas/farmacologia , Permeabilidade/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fusarium/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mucina-1/genética , Mucina-1/metabolismo , Mucina-2/genética , Mucina-2/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Suínos , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo
16.
Gastroenterology ; 157(6): 1544-1555.e3, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31473225

RESUMO

BACKGROUND & AIMS: Sperm flagellar 1 (also called CLAMP) is a microtubule-associated protein that regulates microtubule dynamics and planar cell polarity in multi-ciliated cells. We investigated the localization and function of sperm flagellar 1, or CLAMP, in human intestinal epithelia cells (IECs). METHODS: We performed studies with SKCO-15 and human intestinal enteroids established from biopsies from different intestinal segments (duodenal, jejunum, ileal, and colon) of a single donor. Enteroids were induced to differentiation after incubation with growth factors. The distribution of endogenous CLAMP in IECs was analyzed by immunofluorescence microscopy using total internal reflection fluorescence-ground state depletion and confocal microscopy. CLAMP localization was followed during the course of intestinal epithelial cell polarization as cells progressed from flat to compact, confluent monolayers. Protein interactions with endogenous CLAMP were determined in SKCO-15 cells using proximity ligation assays and co-immunoprecipitation. CLAMP was knocked down in SKCO-15 monolayers using small hairpin RNAs and cells were analyzed by immunoblot and immunofluorescence microscopy. The impact of CLAMP knock-down in migrating SKCO-15 cells was assessed using scratch-wound assays. RESULTS: CLAMP bound to actin and apical junctional complex proteins but not microtubules in IECs. In silico analysis predicted the calponin-homology domain of CLAMP to contain conserved amino acids required for actin binding. During IEC polarization, CLAMP distribution changed from primarily basal stress fibers and cytoplasm in undifferentiated cells to apical membranes and microvilli in differentiated monolayers. CLAMP accumulated in lamellipodia and filopodia at the leading edge of migrating cells in association with actin. CLAMP knock-down reduced the number of filopodia, perturbed filopodia polarity, and altered the organization of actin filaments within lamellipodia. CONCLUSIONS: CLAMP is an actin-binding protein, rather than a microtubule-binding protein, in IECs. CLAMP distribution changes during intestinal epithelial cell polarization, regulates the formation of filopodia, and appears to assist in the organization of actin bundles within lamellipodia of migrating IECs. Studies are needed to define the CLAMP domains that interact with actin and whether its loss from IECs affects intestinal function.


Assuntos
Actinas/metabolismo , Movimento Celular , Mucosa Intestinal/citologia , Proteínas dos Microfilamentos/metabolismo , Pseudópodes/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Colo/citologia , Colo/metabolismo , Células Epiteliais , Humanos , Mucosa Intestinal/metabolismo , Microtúbulos/metabolismo
17.
Mar Drugs ; 17(8)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370332

RESUMO

The effect of collagen peptides (CPs) in intestinal mucosal protection has been approved in both cell and animal models. However, its structure-activity relationship and efficient peptide sequences are unclear, which hinders the in-depth study of its action mechanism and relative nutraceuticals and pharmaceuticals development. In this work, size exclusion chromatography, cation-exchange chromatography, and RP-HPLC were used to separate Alaska pollock skin-derived collagen hydrolysates based on their molecular weight, charge property, and hydrophobicity. The intestinal epithelial barrier function (IEBF) protective effect of separated peptide fractions were evaluated by tumor necrosis factor (TNF)-α-induced Caco-2 cell model. Results indicated that lower molecular weight (500-1000 Da) and higher hydrophilicity of CPs were related to better IEBF protective effect. Two high-efficiency IEBF protective peptide sequences, GPSGPQGSR and GPSGLLGPK with the corresponding molecular weights of 841.41 Da and 824.38 Da, were subsequently identified by UPLC-QToF-MS/MS. Their IEBF protective ability are comparable or even better than the currently used intestinal health supplements glutamine and arginine. The present findings suggested that the hydrophilic CPs, with molecular weight between 500 Da to 1000 Da, should be preferred in IEBF protective peptides preparation. GPSGPQGSR and GPSGLLGPK might have the potential of being IEBF protective ingredients used in intestinal health supplements and drugs.


Assuntos
Colágeno/farmacologia , Gadiformes , Mucosa Intestinal/efeitos dos fármacos , Peptídeos/farmacologia , Alaska , Animais , Células CACO-2 , Colágeno/química , Colágeno/isolamento & purificação , Suplementos Nutricionais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Permeabilidade/efeitos dos fármacos , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem
18.
Science ; 365(6454): 705-710, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31416964

RESUMO

Steady-state turnover is a hallmark of epithelial tissues throughout adult life. Intestinal epithelial turnover is marked by continuous cell migration, which is assumed to be driven by mitotic pressure from the crypts. However, the balance of forces in renewal remains ill-defined. Combining biophysical modeling and quantitative three-dimensional tissue imaging with genetic and physical manipulations, we revealed the existence of an actin-related protein 2/3 complex-dependent active migratory force, which explains quantitatively the profiles of cell speed, density, and tissue tension along the villi. Cells migrate collectively with minimal rearrangements while displaying dual-apicobasal and front-back-polarity characterized by actin-rich basal protrusions oriented in the direction of migration. We propose that active migration is a critical component of gut epithelial turnover.


Assuntos
Movimento Celular/fisiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Mitose , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Animais , Movimento Celular/genética , Polaridade Celular , Imagem Tridimensional , Mucosa Intestinal/metabolismo , Camundongos Knockout , Modelos Biológicos
19.
Gastroenterology ; 157(6): 1556-1571.e5, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31442438

RESUMO

BACKGROUND & AIMS: It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs. METHODS: We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation. Intestine tissues were collected from Tg(GBS-GFP), Sufuf/f; Wnt1-cre, Ptch1+/-, and Gli3Δ699/Δ699 mice and analyzed by flow cytometry and immunofluorescence for levels of messenger RNAs encoding factors in the hedgehog signaling pathway during differentiation of enteric NCs. Human NC cells (HNK-1+p75NTR+) were derived from IMR90 and UE02302 hPSC lines. hPSCs were incubated with a hedgehog agonist (smoothened agonist [SAG]) and antagonists (cyclopamine) and analyzed for differentiation. hPSC-based innervated colonic organoids were derived from these hPSC lines and analyzed by immunofluorescence and neuromuscular coupling assay for expression of neuronal subtype markers and assessment of the functional maturity of the hPSC-derived neurons, respectively. RESULTS: Single-cell RNA sequencing analysis showed that neural fate acquisition by human and mouse enteric NC cells requires reduced expression of NC- and cell cycle-specific genes and up-regulation of neuronal or glial lineage-specific genes. Activation of the hedgehog pathway was associated with progression of mouse enteric NCs to the more mature state along the neuronal and glial lineage differentiation trajectories. Activation of the hedgehog pathway promoted development of cultured hPSCs into NCs of greater neurogenic potential by activating expression of genes in the neurogenic lineage. The hedgehog agonist increased differentiation of hPSCs into cells of the neuronal lineage by up-regulating expression of GLI2 target genes, including INSM1, NHLH1, and various bHLH family members. The hedgehog agonist increased expression of late neuronal markers and neuronal activities in hPSC-derived neurons. CONCLUSIONS: In enteric NCs from humans and mice, activation of hedgehog signaling promotes differentiation into neurons by promoting cell-state transition, expression of genes in the neurogenic lineage, and functional maturity of enteric neurons.


Assuntos
Diferenciação Celular , Proteínas Hedgehog/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Sistema Nervoso Entérico/citologia , Perfilação da Expressão Gênica/métodos , Proteínas Hedgehog/genética , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/inervação , Masculino , Camundongos , Camundongos Transgênicos , Crista Neural/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos
20.
World J Gastroenterol ; 25(30): 4125-4147, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31435168

RESUMO

The introduction of biologics such as anti-tumor necrosis factor (TNF) monoclonal antibodies followed by anti-integrins has dramatically changed the therapeutic paradigm of inflammatory bowel diseases (IBD). Furthermore, a newly developed anti-p40 subunit of interleukin (IL)-12 and IL-23 (ustekinumab) has been recently approved in the United States for patients with moderate to severe Crohn's disease who have failed treatment with anti-TNFs. However, these immunosuppressive therapeutics which focus on anti-inflammatory mechanisms or immune cells still fail to achieve long-term remission in a significant percentage of patients. This strongly underlines the need to identify novel treatment targets beyond immune suppression to treat IBD. Recent studies have revealed the critical role of intestinal epithelial cells (IECs) in the pathogenesis of IBD. Physical, biochemical and immunologic driven barrier dysfunctions of epithelial cells contribute to the development of IBD. In addition, the recent establishment of adult stem cell-derived intestinal enteroid/organoid culture technology has allowed an exciting opportunity to study human IECs comprising all normal epithelial cells. This long-term epithelial culture model can be generated from endoscopic biopsies or surgical resections and recapitulates the tissue of origin, representing a promising platform for novel drug discovery in IBD. This review describes the advantages of intestinal enteroids/organoids as a research tool for intestinal diseases, introduces studies with these models in IBD, and gives a description of the current status of therapeutic approaches in IBD. Finally, we provide an overview of the current endeavors to identify a novel drug target for IBD therapy based on studies with human enteroids/organoids and describe the challenges in using enteroids/organoids as an IBD model.


Assuntos
Descoberta de Drogas/métodos , Imunossupressores/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Organoides/efeitos dos fármacos , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Biópsia , Avaliação Pré-Clínica de Medicamentos/métodos , Células Epiteliais , Humanos , Imunossupressores/uso terapêutico , Células-Tronco Pluripotentes Induzidas , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Organoides/imunologia , Cultura Primária de Células/métodos
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