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1.
Anticancer Res ; 41(7): 3689-3698, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230168

RESUMO

BACKGROUND/AIM: SPARC-related modular calcium-binding protein 2 (SMOC2), a secreted matricellular protein, is reported to be involved in cancer progression such as cell cycle, angiogenesis, and invasion. In this study, we aimed to investigate the expression of SMOC2 in various gastric lesions and assessed its prognostic value in a large cohort of gastric cancer (GC) patients. PATIENTS AND METHODS: SMOC2 mRNA levels were measured by quantitative real-time PCR using 26 matched fresh-frozen GC samples. SMOC2 protein expression was determined by immunohistochemistry on tissue microarrays including 734 GC specimens and its correlations with clinicopathological features and survival were evaluated. RESULTS: The transcription level of SMOC2 was higher in GC samples compared to normal mucosa (p=0.006). Its expression levels were associated with the intestinal stem cell (ISC) marker, LGR5, but there were no correlations with EPHB2 and OLFM4 or the candidate cancer stem cell markers CD133 and CD44. SMOC2 expression was significantly increased in the intestinal metaplasia and was further increased in gastric adenomas and early gastric cancers (EGC). In total, 34% of GCs were positive for SMOC2, and SMOC2 positivity was higher in old (p=0.001) and male (p<0.001) patients, and in well-differentiated GC (p<0.001). SMOC2 expression had a negative association with perineural invasion (p<0.001) and tumor stage (p<0.001). In survival analysis, SMOC2-positive GC patients had much better clinical outcomes in overall survival rates (p<0.001) compared to SMOC2-negative GC patients. The prognostic impact of SMOC2 remained significant both in intestinal (p<0.001) and diffuse-type GC (p<0.001). Remarkably, a multivariate analysis demonstrated SMOC2 as an independent prognostic marker [hazard ratio (HR)=0.732, p=0.045] along with venous invasion (p=0.012), tumor stage (p<0.001) and CDX2 (p=0.028). CONCLUSION: Our results suggest that SMOC2 can be a prognostic marker for better clinical outcomes in GC.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Idoso , Feminino , Humanos , Mucosa Intestinal/patologia , Masculino , Estadiamento de Neoplasias/métodos , Células-Tronco Neoplásicas/patologia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
2.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198897

RESUMO

The introduction of metallic nanoparticles (mNPs) into the diet is a matter of concern for human health. In particular, their effect on the gastrointestinal tract may potentially lead to the increased passage of gluten peptides and the activation of the immune response. In consequence, dietary mNPs could play a role in the increasing worldwide celiac disease (CeD) incidence. We evaluated the potential synergistic effects that peptic-tryptic-digested gliadin (PT) and the most-used food mNPs may induce on the intestinal mucosa. PT interaction with mNPs and their consequent aggregation was detected by transmission electron microscopy (TEM) analyses and UV-Vis spectra. In vitro experiments on Caco-2 cells proved the synergistic cytotoxic effect of PT and mNPs, as well as alterations in the monolayer integrity and tight junction proteins. Exposure of duodenal biopsies to gliadin plus mNPs triggered cytokine production, but only in CeD biopsies. These results suggest that mNPs used in the food sector may alter intestinal homeostasis, thus representing an additional environmental risk factor for the development of CeD.


Assuntos
Doença Celíaca/dietoterapia , Dieta , Glutens/metabolismo , Nanopartículas/uso terapêutico , Biópsia , Células CACO-2 , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Homeostase/imunologia , Humanos , Imunidade/efeitos dos fármacos , Imunidade/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Nanopartículas/metabolismo , Triticum/efeitos adversos
3.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204534

RESUMO

Leaky gut is a condition of increased paracellular permeability of the intestine due to compromised tight junction barriers. In recent years, this affliction has drawn the attention of scientists from different fields, as a myriad of studies prosecuted it to be the silent culprit of various immune diseases. Due to various controversies surrounding its culpability in the clinic, approaches to leaky gut are restricted in maintaining a healthy lifestyle, avoiding irritating factors, and practicing alternative medicine, including the consumption of supplements. In the current study, we investigate the tight junction-modulating effects of processed Aloe vera gel (PAG), comprising 5-400-kD polysaccharides as the main components. Our results show that oral treatment of 143 mg/kg PAG daily for 10 days improves the age-related leaky gut condition in old mice, by reducing their individual urinal lactulose/mannitol (L/M) ratio. In concordance with in vivo experiments, PAG treatment at dose 400 µg/mL accelerated the polarization process of Caco-2 monolayers. The underlying mechanism was attributed to enhancement in the expression of intestinal tight junction-associated scaffold protein zonula occludens (ZO)-1 at the translation level. This was induced by activation of the MAPK/ERK signaling pathway, which inhibits the translation repressor 4E-BP1. In conclusion, we propose that consuming PAG as a complementary food has the potential to benefit high-risk patients.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Preparações de Plantas/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Animais , Biomarcadores , Linhagem Celular , Permeabilidade da Membrana Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Camundongos , Modelos Biológicos , Transdução de Sinais , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo
4.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208517

RESUMO

Superoxide dismutase 3 (SOD3), also known as extracellular superoxide dismutase, is an enzyme that scavenges reactive oxygen species (ROS). It has been reported that SOD3 exerts anti-inflammatory abilities in several immune disorders. However, the effect of SOD3 and the underlying mechanism in inflammatory bowel disease (IBD) have not been uncovered. Therefore, in the present study, we investigated whether SOD3 can protect intestinal cells or organoids from inflammation-mediated epithelial damage. Cells or mice were treated with SOD3 protein or SOD3-transduced mesenchymal stem cells (MSCs). Caco-2 cells or intestinal organoids stimulated with pro-inflammatory cytokines were used to evaluate the protective effect of SOD3 on epithelial junctional integrity. Dextran sulfate sodium (DSS)-induced colitis mice received SOD3 or SOD3-transduced MSCs (SOD3-MSCs), and were assessed for severity of disease and junctional protein expression. The activation of the mitogen-activated protein kinase (MAPK) pathway and elevated expression of cytokine-encoding genes decreased in TNF-α-treated Caco-2 cells or DSS-induced colitis mice when treated with SOD3 or SOD3-MSCs. Moreover, the SOD3 supply preserved the expression of tight junction (ZO-1, occludin) or adherence junction (E-cadherin) proteins when inflammation was induced. SOD3 also exerted a protective effect against cytokine- or ROS-mediated damage to intestinal organoids. These results indicate that SOD3 can effectively alleviate enteritis symptoms by maintaining the integrity of epithelial junctions and regulating inflammatory- and oxidative stress.


Assuntos
Colite/etiologia , Colite/metabolismo , Mucosa Intestinal/metabolismo , Células-Tronco Mesenquimais/metabolismo , Superóxido Dismutase/genética , Junções Íntimas/metabolismo , Animais , Biomarcadores , Células CACO-2 , Colite/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Junções Íntimas/patologia
5.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073605

RESUMO

BACKGROUND: Reduction of the Sphingosine-1-phosphate (S1P) degrading enzyme S1P lyase 1 (SGPL1) initiates colorectal cancer progression with parallel loss of colon function in mice. We aimed to investigate the effect of SGPL1 knockout on the stem cell niche in these mice. METHODS: We performed immunohistochemical and multi-fluorescence imaging on tissue sections of wildtype and SGPL1 knockout colons under disease conditions. Furthermore, we generated SGPL1 knockout DLD-1 cells (SGPL1-/-M.Ex1) using CRISPR/Cas9 and characterized cell cycle and AKT signaling pathway via Western blot, immunofluorescence, and FACS analysis. RESULTS: SGPL1 knockout mice were absent of anti-Ki-67 staining in the stem cell niche under disease conditions. This was accompanied by an increase of the negative cell cycle regulator FOXO3 and attenuation of CDK2 activity. SGPL1-/-M.Ex1 cells show a similar FOXO3 increase but no arrest of proliferation, although we found a suppression of the PDK1/AKT signaling pathway, a prolonged G1-phase, and reduced stem cell markers. CONCLUSIONS: While already established colon cancer cells find escape mechanisms from cell cycle arrest, in vivo SGPL1 knockout in the colon stem cell niche during progression of colorectal cancer can contribute to cell cycle quiescence. Thus, we propose a new function of the S1P lyase 1 in stemness.


Assuntos
Aldeído Liases/metabolismo , Senescência Celular , Proteína Forkhead Box O3/metabolismo , Mucosa Intestinal/metabolismo , Antígeno Ki-67/metabolismo , Células-Tronco/metabolismo , Aldeído Liases/genética , Animais , Linhagem Celular Tumoral , Proteína Forkhead Box O3/genética , Humanos , Antígeno Ki-67/genética , Camundongos , Camundongos Knockout
6.
Clin Transl Gastroenterol ; 12(6): e00348, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34092777

RESUMO

INTRODUCTION: Patients with community-acquired pneumonia display enhanced levels of lipopolysaccharides (LPS) compared with controls, suggesting that low-grade endotoxemia may be implicated in vascular disturbances. It is unknown whether this occurs in patients with coronavirus 2019 (COVID-19) and its impact on thrombotic complications. METHODS: We measured serum levels of zonulin, a marker of gut permeability, LPS, and D-dimer in 81 patients with COVID-19 and 81 healthy subjects; the occurrence of thrombotic events in COVID-19 during the intrahospital stay was registered. RESULTS: Serum LPS and zonulin were higher in patients with COVID-19 than in control subjects and, in COVID-19, significantly correlated (R = 0.513; P < 0.001). Among the 81 patients with COVID-19, 11 (14%) experienced thrombotic events in the arterial (n = 5) and venous circulation (n = 6) during a median follow-up of 18 days (interquartile range 11-27 days). A logistic regression analysis showed that LPS (P = 0.024) and D-dimer (P = 0.041) independently predicted thrombotic events. DISCUSSION: The study reports that low-grade endotoxemia is detectable in patients with COVID-19 and is associated with thrombotic events. The coexistence of low-grade endotoxemia with enhanced levels of zonulin may suggest enhanced gut permeability as an underlying mechanism.


Assuntos
COVID-19 , Endotoxemia , Haptoglobinas/metabolismo , Mucosa Intestinal , Precursores de Proteínas/metabolismo , SARS-CoV-2 , Trombose , Biomarcadores/sangue , COVID-19/sangue , COVID-19/complicações , COVID-19/fisiopatologia , Correlação de Dados , Endotoxemia/diagnóstico , Endotoxemia/metabolismo , Endotoxemia/virologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virologia , Lipopolissacarídeos/análise , Masculino , Pessoa de Meia-Idade , Permeabilidade , Pneumonia Viral/diagnóstico , Pneumonia Viral/etiologia , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Trombose/sangue , Trombose/diagnóstico , Trombose/etiologia
7.
Toxins (Basel) ; 13(6)2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073248

RESUMO

Plant materials can be contaminated with Fusarium mycotoxins and their derivatives, whose toxic effects on humans and animals may remain subclinical. Zearalenone (ZEN), a low-molecular-weight compound, is produced by molds in crop plants as a secondary metabolite. The objective of this study will be to analyze the in vivo correlations between very low monotonic doses of ZEN (5, 10, and 15 µg ZEN/kg body weight-BW for 42 days) and the carryover of this mycotoxin and its selected metabolites from the intestinal contents to the intestinal walls, the mRNA expression of estrogen receptor alfa (ERα) and estrogen receptor beta (ERß) genes, and the mRNA expression of genes modulating selected colon enzymes (CYP1A1 and GSTP1) in the intestinal mucosa of pre-pubertal gilts. An in vivo experiment will be performed on 60 clinically healthy animals with initial BW of 14.5 ± 2 kg. The gilts will be randomly divided into a control group (group C, n = 15) and three experimental groups (group ZEN5, group ZEN10, and group ZEN15; n = 15). Group ZEN5 will be administered per os 5 µg ZEN/kg BW (MABEL), group ZEN10-10 µg ZEN/kg BW (NOAEL), and group ZEN15-15 µg ZEN/kg BW (low LOAEL). In each group, five animals will be euthanized on analytical dates 1 (exposure day 7), 2 (exposure day 21), and 3 (exposure day 42). Samples for in vitro analyses will be collected from an intestinal segment resected from the following regions: the third (horizontal) part of the duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, and descending colon. The experimental material will be collected under special conditions, and it will be transported to specialist laboratories where samples will be obtained for further analyses.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Receptores de Estrogênio/genética , Zearalenona/toxicidade , Animais , Citocromo P-450 CYP1A1/genética , Feminino , Glutationa Transferase/genética , Mucosa Intestinal/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Suínos
8.
Clin Transl Gastroenterol ; 12(6): e00348, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: covidwho-1259760

RESUMO

INTRODUCTION: Patients with community-acquired pneumonia display enhanced levels of lipopolysaccharides (LPS) compared with controls, suggesting that low-grade endotoxemia may be implicated in vascular disturbances. It is unknown whether this occurs in patients with coronavirus 2019 (COVID-19) and its impact on thrombotic complications. METHODS: We measured serum levels of zonulin, a marker of gut permeability, LPS, and D-dimer in 81 patients with COVID-19 and 81 healthy subjects; the occurrence of thrombotic events in COVID-19 during the intrahospital stay was registered. RESULTS: Serum LPS and zonulin were higher in patients with COVID-19 than in control subjects and, in COVID-19, significantly correlated (R = 0.513; P < 0.001). Among the 81 patients with COVID-19, 11 (14%) experienced thrombotic events in the arterial (n = 5) and venous circulation (n = 6) during a median follow-up of 18 days (interquartile range 11-27 days). A logistic regression analysis showed that LPS (P = 0.024) and D-dimer (P = 0.041) independently predicted thrombotic events. DISCUSSION: The study reports that low-grade endotoxemia is detectable in patients with COVID-19 and is associated with thrombotic events. The coexistence of low-grade endotoxemia with enhanced levels of zonulin may suggest enhanced gut permeability as an underlying mechanism.


Assuntos
COVID-19 , Endotoxemia , Haptoglobinas/metabolismo , Mucosa Intestinal , Precursores de Proteínas/metabolismo , SARS-CoV-2 , Trombose , Biomarcadores/sangue , COVID-19/sangue , COVID-19/complicações , COVID-19/fisiopatologia , Correlação de Dados , Endotoxemia/diagnóstico , Endotoxemia/metabolismo , Endotoxemia/virologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virologia , Lipopolissacarídeos/análise , Masculino , Pessoa de Meia-Idade , Permeabilidade , Pneumonia Viral/diagnóstico , Pneumonia Viral/etiologia , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Trombose/sangue , Trombose/diagnóstico , Trombose/etiologia
9.
FASEB J ; 35(7): e21699, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34151459

RESUMO

FUT2, a protein that uses l-fucose to mediate fucosylation of intestinal epithelial cells, is one of the detected gene variants in IBD patients. We aimed to investigate whether exogenous l-fucose could be an enteral nutritional supplement to protect intestinal barrier function. The effect of l-fucose on the restoration of epithelial barrier function in both the DSS-induced colitis mouse model and LPS-stimulated Caco-2 cells was investigated, and the impact on fucosylation of epithelial cells was examined. The severity of DSS-induced colitis was significantly reduced by l-fucose. Restoration of epithelial barrier function by l-fucose was detected. Direct l-fucose-mediated protection of tight junctions was observed in Caco-2 cells. Moreover, exogenous l-fucose promoted the exogenous metabolic pathway of l-fucose, and fucosylation of epithelial cells both in vivo and in vitro. Moreover, knockout of the FUT2 gene restrained fucosylation and the protective effect of l-fucose on barrier function. The severity of colitis was not improved by l-fucose in Fut2 knockout mice. Therefore we conclude that exogenous l-fucose protects intestinal barrier function and relieves intestinal inflammation via upregulation of FUT2-mediated fucosylation of intestinal epithelial cells.


Assuntos
Colite/prevenção & controle , Células Epiteliais/efeitos dos fármacos , Fucose/farmacologia , Fucosiltransferases/fisiologia , Inflamação/prevenção & controle , Mucosa Intestinal/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Sulfato de Dextrana/toxicidade , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
10.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067987

RESUMO

Intraepithelial lymphocytes (IEL) are widely distributed within the small intestinal epithelial cell (IEC) layer and represent one of the largest T cell pools of the body. While implicated in the pathogenesis of intestinal inflammation, detailed insight especially into the cellular cross-talk between IELs and IECs is largely missing in part due to lacking methodologies to monitor this interaction. To overcome this shortcoming, we employed and validated a murine IEL-IEC (organoids) ex vivo co-culture model system. Using livecell imaging we established a protocol to visualize and quantify the spatio-temporal migratory behavior of IELs within organoids over time. Applying this methodology, we found that IELs lacking CD103 (i.e., integrin alpha E, ITGAE) surface expression usually functioning as a retention receptor for IELs through binding to E-cadherin (CD324) expressing IECs displayed aberrant mobility and migration patterns. Specifically, CD103 deficiency affected the ability of IELs to migrate and reduced their speed during crawling within organoids. In summary, we report a new technology to monitor and quantitatively assess especially migratory characteristics of IELs communicating with IEC ex vivo. This approach is hence readily applicable to study the effects of targeted therapeutic interventions on IEL-IEC cross-talk.


Assuntos
Antígenos CD/metabolismo , Movimento Celular , Processamento de Imagem Assistida por Computador/métodos , Cadeias alfa de Integrinas/metabolismo , Mucosa Intestinal/metabolismo , Linfócitos Intraepiteliais/metabolismo , Organoides/metabolismo , Linfócitos T/fisiologia , Animais , Técnicas de Cocultura , Imunofluorescência , Mucosa Intestinal/citologia , Linfócitos Intraepiteliais/citologia , Camundongos , Organoides/citologia , Análise Espaço-Temporal
11.
Nat Commun ; 12(1): 3339, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099655

RESUMO

The intestinal epithelium is a complex structure that integrates digestive, immunological, neuroendocrine, and regenerative functions. Epithelial homeostasis is maintained by a coordinated cross-talk of different epithelial cell types. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here we show that the intestine-enriched miR-802 is a central regulator of intestinal epithelial cell proliferation, Paneth cell function, and enterocyte differentiation. Genetic ablation of mir-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function and intestinal epithelial proliferation. These effects are mediated in part through derepression of the miR-802 target Tmed9, a modulator of Wnt and lysozyme/defensin secretion in Paneth cells, and the downstream Wnt signaling components Fzd5 and Tcf4. Mutant Tmed9 mice harboring mutations in miR-802 binding sites partially recapitulate the augmented Paneth cell function of mice lacking miR-802. Our study demonstrates a broad miR-802 network that is important for the integration of signaling pathways of different cell types controlling epithelial homeostasis in the small intestine.


Assuntos
Diferenciação Celular/fisiologia , Enterócitos/metabolismo , Intestino Delgado/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Celulas de Paneth/metabolismo , Animais , Proliferação de Células , Feminino , Receptores Frizzled/metabolismo , Expressão Gênica , Células HEK293 , Homeostase/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Intestinos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Salmonella typhimurium , Fator de Transcrição 4/metabolismo , Transcriptoma , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Via de Sinalização Wnt
12.
Nat Commun ; 12(1): 3318, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083536

RESUMO

Dormancy, a reversible quiescent cellular state characterized by greatly reduced metabolic activity, protects from genetic damage, prolongs survival and is crucial for tissue homeostasis and cellular response to injury or transplantation. Dormant cells have been characterized in many tissues, but their identification, isolation and characterization irrespective of tissue of origin remains elusive. Here, we develop a live cell ratiometric fluorescent Optical Stem Cell Activity Reporter (OSCAR) based on the observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. Using the small intestinal crypt as a model, OSCAR reveals in real time the dynamics of dormancy induction and cellular differentiation in vitro, and allows the identification and isolation of several populations of transcriptionally diverse OSCARhigh and OSCARlow intestinal epithelial cell states in vivo. In particular, this reporter is able to identify a dormant OSCARhigh cell population in the small intestine. OSCAR therefore provides a tool for a better understanding of dormant stem cell biology.


Assuntos
RNA Polimerase II/metabolismo , Fase de Repouso do Ciclo Celular/fisiologia , Animais , Separação Celular , Quinase 9 Dependente de Ciclina/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Transcrição Genética
13.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069352

RESUMO

Ulcerative colitis (UC), a severe chronic disease with unclear etiology that is associated with increased risk for colorectal cancer, is accompanied by dysregulation of cytokines. Epstein-Barr virus-induced gene 3 (EBI3) encodes a subunit in the unique heterodimeric IL-12 cytokine family of either pro- or anti-inflammatory function. After having recently demonstrated that upregulation of EBI3 by histone acetylation alleviates disease symptoms in a dextran sulfate sodium (DSS)-treated mouse model of chronic colitis, we now aimed to examine a possible further epigenetic regulation of EBI3 by DNA methylation under inflammatory conditions. Treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC) and TNFα led to synergistic upregulation of EBI3 in human colon epithelial cells (HCEC). Use of different signaling pathway inhibitors indicated NFκB signaling was necessary and proportional to the synergistic EBI3 induction. MALDI-TOF/MS and HPLC-ESI-MS/MS analysis of DAC/TNFα-treated HCEC identified IL-12p35 as the most probable binding partner to form a functional protein. EBI3/IL-12p35 heterodimers (IL-35) induce their own gene upregulation, something that was indeed observed in HCEC cultured with media from previously DAC/TNFα-treated HCEC. These results suggest that under inflammatory and demethylating conditions the upregulation of EBI3 results in the formation of anti-inflammatory IL-35, which might be considered as a therapeutic target in colitis.


Assuntos
Colite Ulcerativa/genética , Interleucinas/genética , Interleucinas/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Linhagem Celular , Colite/genética , Colo/patologia , Metilação de DNA/genética , Epigênese Genética/genética , Expressão Gênica/genética , Humanos , Interleucina-12/metabolismo , Mucosa Intestinal/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/genética , Espectrometria de Massas em Tandem/métodos , Fator de Necrose Tumoral alfa/metabolismo
14.
Phytomedicine ; 88: 153589, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111617

RESUMO

BACKGROUND: Irritable bowel syndrome (IBS) is a functional bowel disorder, in which recurrent abdominal pain is associated with defecation or a change in bowel habits. STW 5-II is a combination of six medicinal herbs with a clinically proven efficacy in managing IBS. AIM: This study aims to establish an in vitro IBS model using mouse intestinal organoids and to explore the anti-inflammatory and tight junction protective activities of the multi-herbal preparation STW 5-II. METHODS: Intestinal organoids were cultured in 1:1 Matrigel™ and medium domes. Inflammation and tight junction disruption were induced by a cocktail of cytokines (TNFα, IFNγ, IL-1ß, IL-6) and bacterial proteins (LPS, flagellin). Organoids were treated with different concentrations of STW 5-II, and its multi-target activity was assessed using microarray analyses, RT-qPCR, immunofluorescence, western blot, immunohistochemistry, and a FITC permeability assay. In addition, we analyzed the expression of pNF-κB, pSTAT1, iNOS and ZO-1. In silico analyses were conducted to predict and identify the active components that may be responsible in mediating the multi-target anti-inflammatory activity of STW 5-II. RESULTS: An organoid based IBS model was successfully established. STW 5-II effectively reduced the cytokines-induced overexpression of the pro-inflammatory mediators pNF-κB, pSTAT1 and iNOS. Moreover, STW 5-II attenuated cytokine-mediated downregulation of the tight junction protein, ZO-1. This finding was confirmed by a FITC permeability assay. In silico analyses revealed a promising inhibitory activity of some isolated compounds from STW 5-II against NF-κB, STAT1 and iNOS. CONCLUSION: STW 5-II possesses multiple anti-inflammatory as well as tight junction protective activities that could explain its clinically proven efficacy in managing IBS symptoms.


Assuntos
Anti-Inflamatórios/farmacologia , Intestinos/efeitos dos fármacos , Síndrome do Intestino Irritável/tratamento farmacológico , Extratos Vegetais/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Simulação por Computador , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Síndrome do Intestino Irritável/etiologia , Camundongos , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , Organoides/metabolismo , Organoides/fisiopatologia , Extratos Vegetais/química , Fator de Transcrição STAT1/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
15.
Nat Commun ; 12(1): 3105, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050144

RESUMO

Environmental factors, mucosal permeability and defective immunoregulation drive overactive immunity to a subset of resident intestinal bacteria that mediate multiple inflammatory conditions. GUT-103 and GUT-108, live biotherapeutic products rationally designed to complement missing or underrepresented functions in the dysbiotic microbiome of IBD patients, address upstream targets, rather than targeting a single cytokine to block downstream inflammation responses. GUT-103, composed of 17 strains that synergistically provide protective and sustained engraftment in the IBD inflammatory environment, prevented and treated chronic immune-mediated colitis. Therapeutic application of GUT-108 reversed established colitis in a humanized chronic T cell-mediated mouse model. It decreased pathobionts while expanding resident protective bacteria; produced metabolites promoting mucosal healing and immunoregulatory responses; decreased inflammatory cytokines and Th-1 and Th-17 cells; and induced interleukin-10-producing colonic regulatory cells, and IL-10-independent homeostatic pathways. We propose GUT-108 for treating and preventing relapse for IBD and other inflammatory conditions characterized by unbalanced microbiota and mucosal permeability.


Assuntos
Bactérias/metabolismo , Colite/microbiologia , Colite/terapia , Citocinas/metabolismo , Disbiose/microbiologia , Microbioma Gastrointestinal , Vida Livre de Germes , Animais , Bactérias/genética , Ácidos e Sais Biliares/metabolismo , Colite/imunologia , Modelos Animais de Doenças , Disbiose/terapia , Fezes/microbiologia , Microbioma Gastrointestinal/imunologia , Microbioma Gastrointestinal/fisiologia , Vida Livre de Germes/imunologia , Vida Livre de Germes/fisiologia , Homeostase , Humanos , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
16.
Diagn Pathol ; 16(1): 40, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: covidwho-1216913

RESUMO

AIMS: Patients with COVID-19 can also have enteric symptoms. Here we analyzed the histopathology of intestinal detachment tissue from a patient with COVID-19. METHODS: The enteric tissue was examined by hematoxylin & eosin stain, PAS (Periodic acid-Schiff) staining, Gram staining, Ziehl-Neelsen stain and Grocott's Methenamine Silver (GMS) Stain. The distribution of CD3, CD4, CK20 and CD68, cytomegalovirus (CMV) and Herpes Simplex Virus (HSV) antigen were determined by immunohistochemistry. In situ hybridization (ISH) of SARS-CoV-2 and Epstein-Barr virus-encoded small RNA (EBER) were also performed. RESULTS: We observed mucosal epithelium shedding, intestinal mucosal erosion, focal inflammatory necrosis with hemorrhage, massive neutrophil infiltration, macrophage proliferation accompanied by minor lymphocyte infiltration. Fungal spores and gram positive cocci but not mycobacteria tuberculosis were identified. Immunohistochemistry staining showed abundant CD68+ macrophages but few lymphocytes infiltration. HSV, CMV and EBV were negative. ISH of SARS-CoV-2 RNA showed positive signal which mostly overlapped with CD68 positivity. CONCLUSIONS: The in situ detection of SARS-CoV-2 RNA in intestinal macrophages implicates a possible route for gastrointestinal infection. Further study is needed to further characterize the susceptibility of enteric cells to SARS-CoV-2 infection.


Assuntos
COVID-19/patologia , Gastroenteropatias/patologia , Mucosa Intestinal/patologia , Macrófagos/virologia , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Idoso , Biomarcadores/metabolismo , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/microbiologia , Teste para COVID-19 , Gastroenteropatias/diagnóstico , Gastroenteropatias/imunologia , Gastroenteropatias/microbiologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Macrófagos/metabolismo , Masculino
17.
Nat Cell Biol ; 23(5): 485-496, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33972729

RESUMO

Coordination of stem cell function by local and niche-derived signals is essential to preserve adult tissue homeostasis and organismal health. The vasculature is a prominent component of multiple stem cell niches. However, its role in adult intestinal homeostasis remains largely understudied. Here we uncover a previously unrecognised crosstalk between adult intestinal stem cells in Drosophila and the vasculature-like tracheal system, which is essential for intestinal regeneration. Following damage to the intestinal epithelium, gut-derived reactive oxygen species activate tracheal HIF-1α and bidirectional FGF/FGFR signalling, leading to reversible remodelling of gut-associated terminal tracheal cells and intestinal stem cell proliferation following damage. Unexpectedly, reactive oxygen species-induced adult tracheal plasticity involves downregulation of the tracheal specification factor trachealess (trh) and upregulation of IGF2 messenger RNA-binding protein (IGF2BP2/Imp). Our results reveal an intestine-vasculature inter-organ communication programme that is essential to adapt the stem cell response to the proliferative demands of the intestinal epithelium.


Assuntos
Adaptação Fisiológica/fisiologia , Células-Tronco Adultas/metabolismo , Homeostase/fisiologia , Células-Tronco/metabolismo , Animais , Drosophila/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia
18.
Hum Genet ; 140(8): 1143-1156, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33974130

RESUMO

Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic-intestinal and retinal-disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.


Assuntos
Oftalmopatias Hereditárias/genética , Mucosa Intestinal/metabolismo , Síndromes de Malabsorção/genética , Microvilosidades/patologia , Mucolipidoses/genética , Polimorfismo de Nucleotídeo Único , Proteínas Qa-SNARE/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Distrofias Retinianas/genética , Idoso , Idoso de 80 Anos ou mais , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Autopsia , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/patologia , Feminino , Regulação da Expressão Gênica , Homozigoto , Humanos , Mucosa Intestinal/patologia , Síndromes de Malabsorção/metabolismo , Síndromes de Malabsorção/patologia , Camundongos , Camundongos Knockout , Microvilosidades/genética , Microvilosidades/metabolismo , Mucolipidoses/metabolismo , Mucolipidoses/patologia , Fenótipo , Proteínas Qa-SNARE/deficiência , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Distrofias Retinianas/metabolismo , Distrofias Retinianas/patologia , Rodopsinas Sensoriais/genética , Rodopsinas Sensoriais/metabolismo , Sequenciamento Completo do Exoma
19.
Nat Commun ; 12(1): 2886, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001900

RESUMO

The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.


Assuntos
Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Mucosa Intestinal/metabolismo , Rim/metabolismo , Microvilosidades/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Saco Vitelino/metabolismo , Animais , Epitélio/metabolismo , Perfilação da Expressão Gênica/métodos , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Intestinos/ultraestrutura , Rim/ultraestrutura , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Receptores Citoplasmáticos e Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
FASEB J ; 35(6): e21551, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34042222

RESUMO

Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about how cytokines shape these processes. Given that the anti-inflammatory cytokine interleukin (IL)-10 promotes intestinal barrier function, and insufficient IL-10 signaling increases susceptibility to intestinal diseases like inflammatory bowel disease, we hypothesized that IL-10 signaling modulates processes underlying IEC proliferation and differentiation. This was tested using in vivo and in vitro IEC-specific IL-10 receptor 1 (IL-10R1) depletion under homeostatic conditions. Our findings revealed that loss of IL-10R1 drove lineage commitment toward a dominant goblet cell phenotype while decreasing absorptive cell-related features. Diminished IL-10 signaling also significantly elevated IEC proliferation with relatively minor changes to apoptosis. Characterization of signaling pathways upstream of proliferation demonstrated a significant reduction in the Wnt inhibitor, DKK1, increased nuclear localization of ß-catenin, and increased transcripts of the proliferation marker, OLFM4, with IL-10R1 depletion. Phosphorylated STAT3 was nearly completely absent in IL-10R1 knockdown cells and may provide a mechanistic link between our observations and the regulation of these cellular processes. Our results demonstrate a novel role for IL-10 signaling in intestinal mucosal homeostasis by regulating proper balance of proliferation and IEC lineage fate.


Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais/patologia , Células Caliciformes/patologia , Mucosa Intestinal/patologia , Receptores de Interleucina-10/fisiologia , Animais , Apoptose , Células Epiteliais/metabolismo , Feminino , Células Caliciformes/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais
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