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1.
Nat Commun ; 12(1): 2281, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863879

RESUMO

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.


Assuntos
Adenoma/imunologia , Colite/patologia , Neoplasias Colorretais/imunologia , Fibroblastos/imunologia , Interleucina-11/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Adenoma/genética , Adenoma/mortalidade , Adenoma/cirurgia , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/imunologia , Colo/citologia , Colo/imunologia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/cirurgia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Intervalo Livre de Doença , Feminino , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Interleucina-11/genética , Subunidade alfa de Receptor de Interleucina-11/genética , Subunidade alfa de Receptor de Interleucina-11/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/imunologia , Organoides , Cultura Primária de Células , Estudos Retrospectivos , Transcriptoma/imunologia , Microambiente Tumoral/imunologia
2.
Science ; 372(6539)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33859001

RESUMO

The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.


Assuntos
Colo/citologia , Células Caliciformes/fisiologia , Mucosa Intestinal/citologia , Muco/fisiologia , Animais , Diferenciação Celular , Colite/induzido quimicamente , Colite/fisiopatologia , Colite Ulcerativa/patologia , Colite Ulcerativa/fisiopatologia , Colo/fisiologia , Células Caliciformes/citologia , Humanos , Mucosa Intestinal/fisiologia , Intestino Delgado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-ets/genética , Transcriptoma
3.
Arch Microbiol ; 203(3): 1221-1229, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33620523

RESUMO

Salicylic acid, widely distributed in the whole plant kingdom, is a benzoic acid derivative acting as a signal substance in plants, but could be related to differences in cancer incidence, as many herbs and spices contain high amounts. Lactobacillus rhamnosus GG (LGG) is one of the best-known lactic acid bacteria that has been studied for over 30 years. Probiotic and/or commensal bacteria of the human microbiota are known to respond to diet constituents. Therefore, the present study aims at investigating the possible effects of salicylic acid on the probiotic properties of LGG, and in vitro cytotoxic effects of combination of salicylic acid and LGG on human colon and prostate cancer cells. Salicylic acid significantly (p < 0.05) increased co-aggregation of LGG with E. coli (~ twofold) and anti-oxidant properties. Furthermore, it also induced the cytotoxic effects of LGG against human colon cancer cells. These results suggest that interaction of LGG with salicylic acid can exert more probiotic properties.


Assuntos
Lactobacillus rhamnosus/fisiologia , Ácido Salicílico/farmacologia , Simbióticos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colo/citologia , Colo/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Humanos , Masculino , Microbiota/fisiologia , Probióticos/farmacologia , Neoplasias da Próstata/microbiologia
4.
J Vis Exp ; (168)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33616099

RESUMO

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.


Assuntos
Colo/citologia , Colonoscopia/métodos , Biópsia Guiada por Imagem/métodos , Cicatrização , Animais , Colo/patologia , Colo/cirurgia , Camundongos
5.
J Vis Exp ; (168)2021 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-33616118

RESUMO

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.


Assuntos
Diferenciação Celular , Colo/citologia , Células Epiteliais/citologia , Mucosa Intestinal/citologia , Cultura Primária de Células/métodos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL
6.
Nat Rev Cancer ; 21(4): 239-256, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33627798

RESUMO

Cancer is a clonal disorder derived from a single ancestor cell and its progenies that are positively selected by acquisition of 'driver mutations'. However, the evolution of positively selected clones does not necessarily imply the presence of cancer. On the contrary, it has become clear that expansion of these clones in phenotypically normal or non-cancer tissues is commonly seen in association with ageing and/or in response to environmental insults and chronic inflammation. Recent studies have reported expansion of clones harbouring mutations in cancer driver genes in the blood, skin, oesophagus, bronchus, liver, endometrium and bladder, where the expansion could be so extensive that tissues undergo remodelling of an almost entire tissue. The presence of common cancer driver mutations in normal tissues suggests a strong link to cancer development, providing an opportunity to understand early carcinogenic processes. Nevertheless, some driver mutations are unique to normal tissues or have a mutation frequency that is much higher in normal tissue than in cancer, indicating that the respective clones may not necessarily be destined for evolution to cancer but even negatively selected for carcinogenesis depending on the mutated gene. Moreover, tissues that are remodelled by genetically altered clones might define functionalities of aged tissues or modified inflammatory processes. In this Review, we provide an overview of major findings on clonal expansion in phenotypically normal or non-cancer tissues and discuss their biological significance not only in cancer development but also in ageing and inflammatory diseases.


Assuntos
Envelhecimento/genética , Proliferação de Células/genética , Evolução Clonal , Células Clonais/citologia , Neoplasias/genética , Envelhecimento/patologia , Anemia Aplástica/genética , Anemia Aplástica/patologia , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Brônquios/citologia , Brônquios/metabolismo , Carcinogênese , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colo/citologia , Colo/metabolismo , Endométrio/citologia , Endométrio/metabolismo , Esôfago/citologia , Esôfago/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Fígado/citologia , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Metaplasia , Mutação , Neoplasias/patologia , Oncogenes/genética , Pele/citologia , Pele/metabolismo , Estômago/patologia , Urotélio/citologia , Urotélio/metabolismo
7.
Nutrients ; 13(2)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567596

RESUMO

Caffeic acid is one of the most abundant hydroxycinnamic acids in fruits, vegetables, and beverages. This phenolic compound reaches relevant concentrations in the colon (up to 126 µM) where it could come into contact with the intestinal cells and exert its anti-inflammatory effects. The aim of this investigation was to study the capacity of caffeic acid, at plausible concentrations from an in vivo point of view, to modulate mechanisms related to intestinal inflammation. Consequently, we tested the effects of caffeic acid (50-10 µM) on cyclooxygenase (COX)-2 expression and prostaglandin (PG)E2, cytokines, and chemokines (IL-8, monocyte chemoattractant protein-1 -MCP-1-, and IL-6) biosynthesis in IL-1ß-treated human myofibroblasts of the colon, CCD-18Co. Furthermore, the capacity of caffeic acid to inhibit the angiotensin-converting enzyme (ACE) activity, to hinder advanced glycation end product (AGE) formation, as well as its antioxidant, reducing, and chelating activity were also investigated. Our results showed that (i) caffeic acid targets COX-2 and its product PGE2 as well as the biosynthesis of IL-8 in the IL-1ß-treated cells and (ii) inhibits AGE formation, which could be related to (iii) the high chelating activity exerted. Low anti-ACE, antioxidant, and reducing capacity of caffeic acid was also observed. These effects of caffeic acid expands our knowledge on anti-inflammatory mechanisms against intestinal inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Ácidos Cafeicos/farmacologia , Gastroenterite/tratamento farmacológico , Intestinos/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antioxidantes/farmacologia , Quelantes/farmacologia , Quimiocinas/efeitos dos fármacos , Colo/citologia , Colo/efeitos dos fármacos , Ciclo-Oxigenase 2/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Dinoprostona/antagonistas & inibidores , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Humanos , Inflamação , Interleucina-1beta/biossíntese , Intestinos/citologia
8.
Nature ; 592(7852): 99-104, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33627870

RESUMO

The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.


Assuntos
Colo/citologia , Íleo/transplante , Mucosa Intestinal/citologia , Organoides/transplante , Regeneração , Medicina Regenerativa/métodos , Síndrome do Intestino Curto/terapia , Animais , Colo/irrigação sanguínea , Colo/inervação , Colo/cirurgia , Modelos Animais de Doenças , Xenoenxertos , Humanos , Íleo/citologia , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/inervação , Mucosa Intestinal/cirurgia , Masculino , Técnicas de Cultura de Órgãos , Organoides/citologia , Ratos , Ratos Endogâmicos Lew , Síndrome do Intestino Curto/patologia , Síndrome do Intestino Curto/cirurgia
9.
Int J Mol Sci ; 21(24)2020 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-33322729

RESUMO

(1) Background: We characterized a novel animal model with obesity-induced constipation because constipation is rarely known in genetically engineered mice (GEM); (2) Methods: The changes in the constipation parameters and mechanisms were analyzed in CRISPR-Cas9-mediated leptin (Lep) knockout (KO) mice from eight to 24 weeks; (3) Results: Significant constipation phenotypes were observed in the Lep KO mice since 16 weeks old. These mice showed a significant decrease in the gastrointestinal motility, mucosal layer thickness and ability for mucin secretion as well as the abnormal ultrastructure of Lieberkühn crypts in the transverse colon. The density or function of the enteric neurons, intestinal Cajal cells (ICC), smooth muscle cells, and the concentration of gastrointestinal (GI) hormones for the GI motility were remarkably changed in Lep KO mice. The downstream signaling pathway of muscarinic acetylcholine receptors (mAChRs) were activated in Lep KO mice, while the expression of adipogenesis-regulating genes were alternatively reduced in the transverse colon of the same mice; (4) Conclusions: These results provide the first strong evidence that Lep KO mice can represent constipation successfully through dysregulation of the GI motility mediated by myenteric neurons, ICC, and smooth muscle cells in the transverse colon during an abnormal function of the lipid metabolism.


Assuntos
Colo/metabolismo , Constipação Intestinal/metabolismo , Motilidade Gastrointestinal , Leptina/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores Muscarínicos/metabolismo , Adipogenia/genética , Animais , Aquaporina 3/metabolismo , Aquaporinas/metabolismo , Sistemas CRISPR-Cas , Colo/citologia , Colo/patologia , Colo/ultraestrutura , Constipação Intestinal/complicações , Constipação Intestinal/genética , Constipação Intestinal/patologia , Modelos Animais de Doenças , Feminino , Hormônios Gastrointestinais/metabolismo , Motilidade Gastrointestinal/genética , Motilidade Gastrointestinal/fisiologia , Células Intersticiais de Cajal/metabolismo , Leptina/genética , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mucinas/metabolismo , Neurônios/metabolismo , Obesidade/complicações , Obesidade/genética , Transdução de Sinais/genética
10.
PLoS One ; 15(12): e0243499, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33326448

RESUMO

Excessive alcohol consumption is a risk factor associated with colorectal cancer; however, some epidemiological studies have reported that moderate alcohol consumption may not contribute additional risk or may provide a protective effect reducing colorectal cancer risk. Prior research highlights the importance of proliferation, differentiation, and apoptosis as parameters to consider when evaluating colonic cell growth and tumorigenesis. The present study investigated whether chronic low-to-moderate ethanol consumption altered these parameters of colonic cell growth and expression of related genes. Twenty-four nondeprived young adult (109 days old) and 24 nondeprived middle-aged (420 days old) Wistar rats were randomly assigned to an ethanol-exposed or a water control group (n = 12/group). The ethanol group was provided voluntary access to a 20% v/v ethanol solution on alternate days for 13 weeks. Colon tissues were collected for quantitative immunohistochemical analyses of cell proliferation, differentiation and apoptosis using Ki-67, goblet cell and TUNEL, respectively. Gene expression of cyclin D1 (Ccnd1), Cdk2, Cdk4, p21waf1/cip1 (Cdkn1a), E-cadherin (Cdh1) and p53 were determined by quantitative real-time polymerase chain reaction in colonic scraped mucosa. Ethanol treatment resulted in a lower cell proliferation index and proliferative zone, and lower Cdk2 expression in both age groups, as well as trends toward lower Ccnd1 and higher Cdkn1a expression. Cell differentiation was modestly but significantly reduced by ethanol treatment only in older animals. Overall, older rats showed decreases in apoptosis and gene expression of Cdk4, Cdh1, and p53 compared to younger rats, but there was no observed effect of ethanol exposure on these measures. These findings suggest that low-to-moderate ethanol consumption improves at least one notable parameter in colonic tumorigenesis (cell proliferation) and associated gene expression regardless of age, however, selectively decreased cell differentiation among older subjects.


Assuntos
Etanol/farmacologia , Expressão Gênica/efeitos dos fármacos , Envelhecimento , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colo/citologia , Colo/metabolismo , Colo/patologia , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Masculino , Ratos , Ratos Wistar , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
11.
PLoS One ; 15(10): e0239601, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33112876

RESUMO

APC mutations drive human colorectal cancer (CRC) development. A major contributing factor is colonic stem cell (SC) overpopulation. But, the mechanism has not been fully identified. A possible mechanism is the dysregulation of neuroendocrine cell (NEC) maturation by APC mutations because SCs and NECs both reside together in the colonic crypt SC niche where SCs mature into NECs. So, we hypothesized that sequential inactivation of APC alleles in human colonic crypts leads to progressively delayed maturation of SCs into NECs and overpopulation of SCs. Accordingly, we used quantitative immunohistochemical mapping to measure indices and proportions of SCs and NECs in human colon tissues (normal, adenomatous, malignant), which have different APC-zygosity states. In normal crypts, many cells staining for the colonic SC marker ALDH1 co-stained for chromogranin-A (CGA) and other NEC markers. In contrast, in APC-mutant tissues from familial adenomatous polyposis (FAP) patients, the proportion of ALDH+ SCs progressively increased while NECs markedly decreased. To explain how these cell populations change in FAP tissues, we used mathematical modelling to identify kinetic mechanisms. Computational analyses indicated that APC mutations lead to: 1) decreased maturation of ALDH+ SCs into progenitor NECs (not progenitor NECs into mature NECs); 2) diminished feedback signaling by mature NECs. Biological experiments using human CRC cell lines to test model predictions showed that mature GLP-2R+ and SSTR1+ NECs produce, via their signaling peptides, opposing effects on rates of NEC maturation via feedback regulation of progenitor NECs. However, decrease in this feedback signaling wouldn't explain the delayed maturation because both progenitor and mature NECs are depleted in CRCs. So the mechanism for delayed maturation must explain how APC mutation causes the ALDH+ SCs to remain immature. Given that ALDH is a key component of the retinoic acid (RA) signaling pathway, that other components of the RA pathway are selectively expressed in ALDH+ SCs, and that exogenous RA ligands can induce ALDH+ cancer SCs to mature into NECs, RA signaling must be attenuated in ALDH+ SCs in CRC. Thus, attenuation of RA signaling explains why ALDH+ SCs remain immature in APC mutant tissues. Since APC mutation causes increased WNT signaling in FAP and we found that sequential inactivation of APC in FAP patient tissues leads to progressively delayed maturation of colonic ALDH+ SCs, the hypothesis is developed that human CRC evolves due to an imbalance between WNT and RA signaling.


Assuntos
Transformação Celular Neoplásica/genética , Colo/citologia , Colo/metabolismo , Neoplasias Colorretais/genética , Genes APC , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Mutação , Somatostatina/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromogranina A/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Retroalimentação Fisiológica , Receptor do Peptídeo Semelhante ao Glucagon 2/metabolismo , Células HCT116 , Células HT29 , Humanos , Camundongos , Modelos Genéticos , Células Neuroendócrinas/citologia , Células Neuroendócrinas/metabolismo , Receptores de Somatostatina/metabolismo , Transdução de Sinais , Nicho de Células-Tronco , Tretinoína/metabolismo , Via de Sinalização Wnt
12.
Cells ; 9(9)2020 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-32932592

RESUMO

Feline coronaviruses (FCoVs) infect both wild and domestic cat populations world-wide. FCoVs present as two main biotypes: the mild feline enteric coronavirus (FECV) and the fatal feline infectious peritonitis virus (FIPV). FIPV develops through mutations from FECV during a persistence infection. So far, the molecular mechanism of FECV-persistence and contributing factors for FIPV development may not be studied, since field FECV isolates do not grow in available cell culture models. In this work, we aimed at establishing feline ileum and colon organoids that allow the propagation of field FECVs. We have determined the best methods to isolate, culture and passage feline ileum and colon organoids. Importantly, we have demonstrated using GFP-expressing recombinant field FECV that colon organoids are able to support infection of FECV, which were unable to infect traditional feline cell culture models. These organoids in combination with recombinant FECVs can now open the door to unravel the molecular mechanisms by which FECV can persist in the gut for a longer period of time and how transition to FIPV is achieved.


Assuntos
Coronavirus Felino/crescimento & desenvolvimento , Peritonite Infecciosa Felina/patologia , Técnicas de Cultura de Órgãos/veterinária , Organoides/crescimento & desenvolvimento , Animais , Gatos , Linhagem Celular , Colo/citologia , Colo/virologia , Coronavirus Felino/genética , Feminino , Células HEK293 , Humanos , Íleo/citologia , Íleo/virologia , Modelos Biológicos , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia
13.
Cell Rep ; 32(1): 107863, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32610043

RESUMO

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is an unprecedented worldwide health problem that requires concerted and global approaches to stop the coronavirus 2019 (COVID-19) pandemic. Although SARS-CoV-2 primarily targets lung epithelium cells, there is growing evidence that the intestinal epithelium is also infected. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of the SARS-CoV-2 life cycle in human intestinal epithelial cells (hIECs). Our results demonstrate that hIECs fully support SARS-CoV-2 infection, replication, and production of infectious de novo virus particles. We found that viral infection elicits an extremely robust intrinsic immune response where interferon-mediated responses are efficient at controlling SARS-CoV-2 replication and de novo virus production. Taken together, our data demonstrate that hIECs are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing to increasing patient viremia and fueling an exacerbated cytokine response.


Assuntos
Betacoronavirus/crescimento & desenvolvimento , Colo/virologia , Células Epiteliais/imunologia , Interferons/imunologia , Mucosa Intestinal/imunologia , Betacoronavirus/imunologia , Células CACO-2 , Linhagem Celular Tumoral , Colo/citologia , Colo/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/virologia , Citocinas/sangue , Células Epiteliais/virologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/virologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/patologia , Replicação Viral/imunologia
14.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 36(1): 90-93, 2020 Jan 28.
Artigo em Chinês | MEDLINE | ID: mdl-32476379

RESUMO

OBJECTIVE: To observe the regulatory effect of 6-Shogaol on Notch signal pathway in colonic epithelial cells of mice with ulcerative colitis. METHODS: Forty Kunming mice were randomly divided into normal group (n=10) and model group (n=30). The model of ulcerative colitis was induced by free drinking of 2% dextran sulfate sodium salt(DSS). After 15 days, the mice were divided into model group, 6-gingerenol group and positive control group with 10 mice in each group. Normal group and model group were treated with normal saline, 6-gingerenol group was treated with 6-Shogaol 100 mg/(kg·d), positive control group was treated with sulfasalazine 100 mg/(kg·d), for 20 days. The histopathological changes of colon were observed, and the expressions of Hes-1 and Math-1protein in colonic epithelial cells were detected by immunofluorescence double labeling method. The expressions of Notch-1, Hes-1 and Math-1 mRNA in colonic epithelial tissue were detected by RT-PCR. The expressions of Notch-1, Hes-1 and Math-1 protein in colonic epithelial tissue was detected by Western blot. RESULTS: Compared with the normal group, the expression of Notch-1 and Hes-1 protein and the relative expression of mRNA in colonic epithelium of model group were significantly increased (P<0.01), while the relative expressions of Math-1 mRNA and protein were decreased significantly (P<0.01). Compared with the model group, the expressions of Notch-1 and Hes-1 protein and the relative expression of mRNA in colonic epithelium of 6-Shogaol group and sulfasalazine group were decreased significantly(P<0.01), while the relative expressions of Math-1 mRNA and protein were increased significantly(P<0.01). CONCLUSION: 6-Shogaol can inhibit the over activation of Notch pathway and regulate the balance of differentiation between colonic epithelialabsorptive cell line and secretory cell line and repair damaged mucosal tissue.


Assuntos
Catecóis/farmacologia , Colite Ulcerativa , Células Epiteliais/efeitos dos fármacos , Transdução de Sinais , Animais , Colo/citologia , Modelos Animais de Doenças , Mucosa Intestinal/patologia , Camundongos , Receptores Notch/metabolismo
15.
PLoS One ; 15(5): e0223344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365104

RESUMO

Stilbenes are a group of chemicals characterized with the presence of 1,2-diphenylethylene. Previously, our group has demonstrated that synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) possesses potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. In this study, the cytoprotective effects of BK3C231 on cellular DNA and mitochondria were investigated in normal human colon fibroblast, CCD-18Co cells. The cells were pretreated with BK3C231 prior to exposure to the carcinogen 4-nitroquinoline 1-oxide (4NQO). BK3C231 was able to inhibit 4NQO-induced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. These new findings elucidate the cytoprotective potential of BK3C231 in human colon fibroblast CCD-18Co cell model which warrants further investigation into its chemopreventive role.


Assuntos
4-Nitroquinolina-1-Óxido/toxicidade , Colo/efeitos dos fármacos , Citoproteção , Dano ao DNA/efeitos dos fármacos , Furanos/farmacologia , Mutagênicos/toxicidade , Estilbenos/farmacologia , Linhagem Celular , Colo/citologia , Reparo do DNA/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Furanos/química , Humanos , Mitocôndrias/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estilbenos/química
16.
PLoS One ; 15(5): e0232934, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428045

RESUMO

AIMS: Much work has been done to find markers of cancer stem cells (CSCs) that distinguish them from the tumor bulk cells and normal cells. Recent CSC research has applied the induced pluripotent stem cell (iPSC) concept. In this study, we investigated the expression of a panel of iPSC markers in primary colon adenocarcinoma (CA)-derived cell lines. MATERIALS AND METHODS: Expression of iPSC markers by CA-derived primary cell lines was interrogated using immunocytochemistry, western blotting and RT-qPCR. The stem cell function of these cells was then assessed in vitro using differentiation and tumorsphere assays. RESULTS: Expression of iPSC markers OCT4, SOX2, NANOG, KLF4 and c-MYC was more widespread in high-grade CA (HGCA) cell lines than low-grade CA (LGCA) cell lines, as demonstrated by western blotting and RT-qPCR. These cells could be induced to differentiate down the three embryonic lineages. Cells derived from HGCA were more capable of forming tumorspheres than those derived from LGCA. EpCAM sorting revealed that a population enriched for EpCAMHigh cells formed larger tumorspheres than EpCAMLow cells. Pluripotency markers, SSEA4 and TRA-1-60, were co-expressed by a small subpopulation of cells that also co-expressed SOX2 in 75% and OCT4 in 50% of the cell lines. CONCLUSIONS: CA-derived primary cell lines contain tumorsphere-forming cells which express key pluripotency genes and can differentiate down 3 embryonic lineages, suggesting a pluripotent CSC-like phenotype. There appear to be two iPSC-like subpopulations, one with high EpCAM expression which forms larger tumorspheres than another with low EpCAM expression. Furthermore, these cells can be characterized based on iPSC marker expression, as we have previously demonstrated in the original CA tumor tissues.


Assuntos
Adenocarcinoma/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Reprogramação Celular/genética , Colo/citologia , Colo/metabolismo , Neoplasias do Colo/metabolismo , Proteínas de Ligação a DNA/análise , Genes Homeobox , Genes myc , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição Kruppel-Like/análise , Proteína Homeobox Nanog/análise , Fator 3 de Transcrição de Octâmero/análise , Cultura Primária de Células , Fatores de Transcrição SOXB1/análise , Fatores de Transcrição/análise
17.
PLoS One ; 15(4): e0231423, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302323

RESUMO

Recent advances in canine intestinal organoids have expanded the option for building a better in vitro model to investigate translational science of intestinal physiology and pathology between humans and animals. However, the three-dimensional geometry and the enclosed lumen of canine intestinal organoids considerably hinder the access to the apical side of epithelium for investigating the nutrient and drug absorption, host-microbiome crosstalk, and pharmaceutical toxicity testing. Thus, the creation of a polarized epithelial interface accessible from apical or basolateral side is critical. Here, we demonstrated the generation of an intestinal epithelial monolayer using canine biopsy-derived colonic organoids (colonoids). We optimized the culture condition to form an intact monolayer of the canine colonic epithelium on a nanoporous membrane insert using the canine colonoids over 14 days. Transmission and scanning electron microscopy revealed a physiological brush border interface covered by the microvilli with glycocalyx, as well as the presence of mucin granules, tight junctions, and desmosomes. The population of stem cells as well as differentiated lineage-dependent epithelial cells were verified by immunofluorescence staining and RNA in situ hybridization. The polarized expression of P-glycoprotein efflux pump was confirmed at the apical membrane. Also, the epithelial monolayer formed tight- and adherence-junctional barrier within 4 days, where the transepithelial electrical resistance and apparent permeability were inversely correlated. Hence, we verified the stable creation, maintenance, differentiation, and physiological function of a canine intestinal epithelial barrier, which can be useful for pharmaceutical and biomedical researches.


Assuntos
Colo/citologia , Células Epiteliais/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Desmossomos/metabolismo , Cães , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Membranas Artificiais , Microvilosidades/fisiologia , Mucinas/metabolismo , Nanoporos , Células-Tronco/citologia , Células-Tronco/metabolismo , Junções Íntimas/metabolismo
18.
PLoS One ; 15(4): e0231942, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32339193

RESUMO

Salmonella enterica serovar Typhimurium is an animal welfare and public health concern due to its ability to parasite livestock and potentially contaminate pork products. To reduce Salmonella shedding and the risk of pork contamination, antibiotic therapy is used and can contribute to antimicrobial resistance. Here we hypothesized that immune system education by the microbiota can play a role in intestinal resilience to infection. We used amoxicillin (15mg/Kg) to modulate the intestinal microbiome of 10 piglets, paired with same age pigs that received a placebo (n = 10) from 0 to 14 days of age. Animals were euthanized at 4-weeks old. Each pig donated colon sections for ex vivo culture (n = 20 explants/pig). Explants were inoculated with S. Typhimurium, PBS or LPS (n = 6 explants/pig/group, plus technical controls). The gut bacteriome was characterized by sequencing of the 16S rRNA at 7, 21 days of age, and upon in vitro culture. Explants response to infection was profiled through high-throughput mRNA sequencing. In vivo antibiotic treatment led to ß-diversity differences between groups at all times (P<0.05), while α-diversity did not differ between amoxicillin and placebo groups on day 21 and at euthanasia (P<0.03 on day 7). Explant microbiomes were not different from in vivo. In vitro challenge with S. Typhimurium led to lower necrosis scores in explants from amoxicillin-treated pigs, when compared to explants placebo-treated pigs (P<0.05). This was coupled with the activation of immune-related pathways in explants from amoxicillin-treated pigs (IL-2 production, NO production, BCR activation), when compared to placebo-treated pigs. In addition, several DNA repair and intestinal wound healing pathways were also only activated in explants from amoxicillin-treated pigs. Taken together, these findings suggest that immune education by the amoxicillin-disturbed microbiota may have contributed to mitigate intestinal lesions following pathogen exposure.


Assuntos
Antibacterianos/farmacologia , Epigênese Genética/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Salmonella typhimurium/patogenicidade , Amoxicilina/farmacologia , Animais , Animais Recém-Nascidos , Bactérias/genética , Bactérias/isolamento & purificação , Colo/citologia , Colo/microbiologia , Colo/patologia , Regulação para Baixo/efeitos dos fármacos , Fezes/microbiologia , Análise de Componente Principal , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Salmonelose Animal/imunologia , Suínos , Doenças dos Suínos/imunologia , Regulação para Cima/efeitos dos fármacos
19.
Vet Microbiol ; 243: 108632, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32273011

RESUMO

Zinc treatment is beneficial for infectious diarrhea or colitis. This study aims to characterize the pathomechanisms of the epithelial barrier dysfunction caused by alpha-hemolysin (HlyA)-expressing Escherichia coli in the colon mucosa and the mitigating effects of zinc ions. We performed Ussing chamber experiments on porcine colon epithelium and infected the tissues with HlyA-producing E. coli. Colon mucosa from piglets was obtained from a feeding trial with defined normal or high dose zinc feeding (pre-conditioning). Additional to the zinc feeding, zinc was added to the luminal compartment of the Ussing chamber. Transepithelial electrical resistance (TER) was measured during the infection of the living tissue and subsequently the tissues were immuno-stained for confocal microscopy. Zinc applied to the luminal compartment was effective in preventing from E. coli-induced epithelial barrier dysfunction in Ussing chamber experiments. In contrast, zinc pre-conditioning of colon mucosae when zinc ions were missing subsequently in the luminal compartment was not sufficient to prevent epithelial barrier impairment during E. coli infection. The pathological changes caused by E. coli HlyA were alterations of tight junction proteins claudin-4 and claudin-5, focal leak formation, and cell exfoliation which reflected the paracellular barrier defect measured by a reduced TER. In microscopic analysis of luminal zinc-treated mucosae these changes were absent. In conclusion, continuous presence of unbound zinc ions in the luminal compartment is essential for the protective action of zinc against E. coli HlyA. This suggests the usage of zinc as therapeutic regimen, while prophylactic intervention by high dietary zinc loads may be less useful.


Assuntos
Colo/efeitos dos fármacos , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Zinco/farmacologia , Ração Animal , Animais , Colo/citologia , Colo/microbiologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/prevenção & controle , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Técnicas de Cultura de Órgãos , Suínos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/patologia
20.
J Vis Exp ; (157)2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32225161

RESUMO

Crohn's disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn's disease. A detailed protocol for the induction of Crohn's disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.


Assuntos
Colo/metabolismo , Doença de Crohn/fisiopatologia , Imunofluorescência/métodos , Receptores Estrogênicos/metabolismo , Animais , Colo/citologia , Humanos , Camundongos
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