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1.
Chem Commun (Camb) ; 56(3): 399-402, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31820751

RESUMO

A combinatorial approach using a one-bead-one-compound method and a screening based on a SOD-activity assay was set up for the discovery of an efficient peptidyl copper complex. The complex exhibited good stability constants, suitable redox potentials and excellent intrinsic activity. This complex was further assayed in cells for its antioxidant properties and showed beneficial effects when cells were subjected to oxidative stress.


Assuntos
Materiais Biocompatíveis/metabolismo , Cobre/química , Peptídeos/química , Sequência de Aminoácidos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Cobre/metabolismo , Células HT29 , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/metabolismo , Superóxido Dismutase/metabolismo
2.
Nat Genet ; 51(12): 1723-1731, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31784729

RESUMO

WNT signaling activates MYC expression in cancer cells. Here we report that this involves an oncogenic super-enhancer-mediated tethering of active MYC alleles to nuclear pores to increase transcript export rates. As the decay of MYC transcripts is more rapid in the nucleus than in the cytoplasm, the oncogenic super-enhancer-facilitated export of nuclear MYC transcripts expedites their escape from the nuclear degradation system in colon cancer cells. The net sum of this process, as supported by computer modeling, is greater cytoplasmic MYC messenger RNA levels in colon cancer cells than in wild type cells. The cancer-cell-specific gating of MYC is regulated by AHCTF1 (also known as ELYS), which connects nucleoporins to the oncogenic super-enhancer via ß-catenin. We conclude that WNT signaling collaborates with chromatin architecture to post-transcriptionally dysregulate the expression of a canonical cancer driver.


Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Genes myc , Fatores de Transcrição/genética , Via de Sinalização Wnt/genética , Colo/citologia , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/fisiologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Processamento Pós-Transcricional do RNA , Fatores de Transcrição/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
3.
Zhonghua Wei Chang Wai Ke Za Zhi ; 22(11): 1095-1100, 2019 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-31770844

RESUMO

Colonic organoids are three-dimensional organotypic cultures of the colonic stem cells or pluripotent stem cells. Its essence is the culture of colonic stem cells or pluripotent stem cells, and their derived intestinal epithelial cells, intestinal endocrine cells and goblet cells in basement membrane extract with specific growth factors. Colonic organoids are comprised of all major types of colonic epithelial cells and represent the architecture and function remarkably similar to those of the colonic epithelium, faithfully recapitulating the functional colonic epithelium ex vivo. As a superior basic experimental model, colonic organoids are representing advantages over conventional cell models and animal models in many aspects, such as high successful rate, short productive cycle, and high consistency with source tissue. Since first reported in 2011, colonic organoids have soon become an important topic in the field of colonic diseases. It has now been applied in the field of physiology of colonic epithelium, infectious diarrhea, ulcerative colitis, regeneration of intestinal injury, and colon tumors. In this review, we summarize the research advances of establishment and application of colonic organoids.


Assuntos
Colo/fisiologia , Mucosa Intestinal/fisiologia , Organoides/fisiologia , Células-Tronco/fisiologia , Animais , Colo/citologia , Humanos , Intestinos
4.
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
5.
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
6.
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
7.
Soft Matter ; 15(36): 7203-7210, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31475279

RESUMO

Cancer metastasis has been believed as a genetically programmed process that is commonly marked by biochemical signals. Here using extracellular matrix control of cellular mechanics, we establish that cellular force threshold can also mark in vitro metastatic phenotypic change and malignant transformation in HCT-8 cell colonies. We observe that for prolonged culture time the HCT-8 cell colonies disperse into individual malignant cells, and the metastatic-like dispersion depends on both cell-seeding gel stiffness and colony size. Cellular force microscopies show that gel stiffness and colony size are also two key parameters that modulate cellular forces, suggesting the correlations between the cellular forces and the metastatic phenotypic change. Using our recently developed biophysical model, we construct an extracellular traction phase diagram in the stiffness-size space, filled with experimental data on the colony behavior. From the phase diagram we identify a phase boundary as a traction force threshold above which the metastatic phenotypic transition occurs and below which the cell colonies remain cohesive. Our finding suggests that the traction threshold can be regarded as an effective mechano-marker for the onset of the metastatic-like dispersion and malignant transformation.


Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Colo/citologia , Matriz Extracelular/metabolismo , Humanos , Mecanotransdução Celular , Modelos Biológicos , Fenótipo , Estresse Mecânico
8.
Biol Sex Differ ; 10(1): 47, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492202

RESUMO

BACKGROUND: Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions. METHODS: Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression. RESULTS: Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance. CONCLUSIONS: Altogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt.


Assuntos
Colo/citologia , Receptor PAR-2/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides/fisiologia , Receptor PAR-2/genética , Caracteres Sexuais
9.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(4): 506-511, 2019 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-31484613

RESUMO

To investigate the expressions of mucosal barrier proteins in colon cell line DLD-1 under hypoxic environment in vitro and its mechanism. Methods After DLD-1 cells were treated separately with hypoxia(l% O2),vitamin D(100 nmol/L),or vitamin D plus hypoxia for 48 hours,the expressions of vitamin D receptor(VDR),tight junction proteins zonula occludens-1(ZO-1),occludin,Claudin-1,and adherent junction protein(E-cadherin)were determined by Western blot.Stable VDR knock-down(Sh-VDR)DLD-1 cell line and control DLD-1 cell line were established by lentivirus package technology and the protein expressions after hypoxia treatment were detected. Results Compared with control group,the expressions of occludin,Claudin-1,and VDR increased significantly after hypoxia treatment(all P<0.001).In addition to the protein expressions of occludin,Claudin-1 and VDR,the expressions of ZO-1 and E-cadherin were also obviously higher in vitamin D plus hypoxia group than in single vitamin D treatment group(all P<0.001).After hypoxia treatment,Sh-VDR cell line showed significantly decreased expressions of ZO-1(P<0.001),occludin(P<0.05),Claudin-1(P<0.01)and E-cadherin(P<0.001)when compared with untreated Sh-VDR cell line. Conclusion VDR acts as a regulator for the expressions of intestinal mucosal barrier proteins under hypoxia environment in DLD-1 colon cell line,indicating that VDR pathway may be another important protective mechanism for gut barrier in low-oxygen environment.


Assuntos
Colo/citologia , Receptores de Calcitriol/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Hipóxia Celular , Linhagem Celular , Claudina-1/metabolismo , Humanos , Ocludina/metabolismo , Junções Íntimas , Vitamina D/farmacologia , Proteína da Zônula de Oclusão-1/metabolismo
10.
J Agric Food Chem ; 67(35): 9831-9839, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31407897

RESUMO

Probiotic lactobacilli and their exopolysaccharides (EPS) are thought to modulate mucosal homeostasis; however, their mechanisms remain elusive. Thus, we tried to clarify the role of exopolysaccharides from Lactobacillus plantarum NCU116 (EPS116) in the intestinal mucosal homeostasis. Our results indicated that EPS116 regulated the colon mucosal healing and homeostasis, enhanced the goblet cell differentiation, and promoted the expression of Muc2 gene in vivo and in vitro. Further experiments showed that EPS116 promoted the expression and phosphorylation of transcription factor c-Jun and facilitated its binding to the promoter of Muc2. Moreover, knocking down c-Jun or inhibiting its function in LS 174T cells treated with EPS116 led to decreased expression of Muc2, implying that EPS116 promoted the colonic mucosal homeostasis and Muc2 expression via c-Jun. Therefore, our study uncovered a novel model where EPS116 enhanced colon mucosal homeostasis by controlling the epithelial cell differentiation and c-Jun/Muc2 signaling.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Lactobacillus plantarum/química , Mucina-2/metabolismo , Polissacarídeos Bacterianos/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Linhagem Celular Tumoral , Colo/citologia , Colo/metabolismo , Colo/fisiopatologia , Homeostase/efeitos dos fármacos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucina-2/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de Sinais/efeitos dos fármacos
11.
World J Gastroenterol ; 25(27): 3503-3526, 2019 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-31367153

RESUMO

Eosinophils are currently regarded as versatile mobile cells controlling and regulating multiple biological pathways and responses in health and disease. These cells store in their specific granules numerous biologically active substances (cytotoxic cationic proteins, cytokines, growth factors, chemokines, enzymes) ready for rapid release. The human gut is the main destination of eosinophils that are produced and matured in the bone marrow and then transferred to target tissues through the circulation. In health the most important functions of gut-residing eosinophils comprise their participation in the maintenance of the protective mucosal barrier and interactions with other immune cells in providing immunity to microbiota of the gut lumen. Eosinophils are closely involved in the development of inflammatory bowel disease (IBD), when their cytotoxic granule proteins cause damage to host tissues. However, their roles in Crohn's disease and ulcerative colitis appear to follow different immune response patterns. Eosinophils in IBD are especially important in altering the structure and protective functions of the mucosal barrier and modulating massive neutrophil influx to the lamina propria followed by transepithelial migration to colorectal mucus. IBD-associated inflammatory process involving eosinophils then appears to expand to the mucus overlaying the internal gut surface. The author hypothesises that immune responses within colorectal mucus as well as ETosis exerted by both neutrophils and eosinophils on the both sides of the colonic epithelial barrier act as additional pathogenetic factors in IBD. Literature analysis also shows an association between elevated eosinophil levels and better colorectal cancer (CRC) prognosis, but mechanisms behind this effect remain to be elucidated. In conclusion, the author emphasises the importance of investigating colorectal mucus in IBD and CRC patients as a previously unexplored milieu of disease-related inflammatory responses.


Assuntos
Colite Ulcerativa/imunologia , Neoplasias Colorretais/imunologia , Doença de Crohn/imunologia , Eosinófilos/imunologia , Mucosa Intestinal/imunologia , Colite Ulcerativa/microbiologia , Colite Ulcerativa/patologia , Colo/citologia , Colo/imunologia , Colo/patologia , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Microbioma Gastrointestinal/imunologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Contagem de Leucócitos
12.
World J Gastroenterol ; 25(27): 3572-3589, 2019 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-31367158

RESUMO

BACKGROUND: Mucosal healing has become a therapeutic goal to achieve stable remission in patients with inflammatory bowel diseases. To achieve this objective, overlapping actions of complex cellular processes, such as migration, proliferation, and differentiation, are required. These events are longitudinally and tightly controlled by numerous factors including a wide range of distinct regulatory proteins. However, the sequence of events associated with colon mucosal repair after colitis and the evolution of the luminal content characteristics during this process have been little studied. AIM: To document the evolution of colon mucosal characteristics during mucosal healing using a mouse model with chemically-induced colitis. METHODS: C57BL/6 male mice were given 3.5% dextran sodium sulfate (DSS) in drinking water for 5 d. They were euthanized 2 (day 7), 5 (day 10), 8 (day 13), and 23 (day 28) d after DSS removal. The colonic luminal environment and epithelial repair processes during the inflammatory flare and colitis resolution were analyzed with reference to a non-DSS treated control group, euthanized at day 0. Epithelial repair events were assessed histo-morphologically in combination with functional permeability tests, expression of key inflammatory and repairing factors, and evaluation of colon mucosa-adherent microbiota composition by 16S rRNA sequencing. RESULTS: The maximal intensity of colitis was concomitant with maximal alterations of intestinal barrier function and histological damage associated with goblet cell depletion in colon mucosa. It was recorded 2 d after termination of the DSS-treatment, followed by a progressive return to values similar to those of control mice. Although signs of colitis were severe (inflammatory cell infiltrate, crypt disarray, increased permeability) and associated with colonic luminal alterations (hyperosmolarity, dysbiosis, decrease in short-chain fatty acid content), epithelial healing processes were launched early during the inflammatory flare with increased gene expression of certain key epithelial repair modulators, including transforming growth factor-ß, interleukin (Il)-15, Il-22, Il-33, and serum amyloid A. Whereas signs of inflammation progressively diminished, luminal colonic environment alterations and microscopic abnormalities of colon mucosa persisted long after colitis induction. CONCLUSION: This study shows that colon repair can be initiated in the context of inflamed mucosa associated with alterations of the luminal environment and highlights the longitudinal involvement of key modulators.


Assuntos
Colite Ulcerativa/imunologia , Colo/patologia , Microbioma Gastrointestinal/imunologia , Mucosa Intestinal/patologia , Regeneração/imunologia , Animais , Movimento Celular , Proliferação de Células , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Colo/citologia , Colo/efeitos dos fármacos , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , RNA Ribossômico 16S
13.
Parasit Vectors ; 12(1): 349, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300009

RESUMO

BACKGROUND: Four species of Strongyloides, Strongyloides felis, Strongyloides planiceps, Strongyloides stercoralis and Strongyloides tumefaciens, have been identified in cats based on morphology and location in the host with limited data on the prevalence and disease potential of these different species. Strongyloides tumefaciens adults are located in colonic nodules while the other three species are in the small intestine. The literature on Strongyloides in cats is scattered and has never been compiled. The aim of this article is to provide a short review of the existing literature on Strongyloides spp. in cats, to describe the pathology of colonic nodules containing Strongyloides sp. seen at necropsies of cats in St. Kitts, West Indies, and to provide the first unequivocal report of zoonotic S. stercoralis in cats based on sequencing analysis of a portion of the cytochrome c oxidase subunit 1 (cox1) gene, and supported by phylogenetic analysis. RESULTS: Colonic nodules containing sections of nematodes, histologically compatible with Strongyloides sp. were seen during necropsy in six cats in St. Kitts, West Indies. Sequencing of the cox1 gene of the mitochondrial DNA extracted from colonic nodules from two of these cats matched sequences of the zoonotic strain of S. stercoralis. CONCLUSIONS: The morphological similarities between S. stercoralis-associated colonic nodules and previous reports of S. tumefaciens, together with the insufficient defining criteria for S. tumefaciens raises questions about the validity of the species. Further sampling and genetic characterization of isolates is needed to understand the species in cats and their zoonotic potential.


Assuntos
Gatos/parasitologia , Colo/patologia , Hiperplasia/parasitologia , Estrongiloidíase/veterinária , Zoonoses/parasitologia , Animais , Colo/citologia , Colo/parasitologia , DNA Mitocondrial/genética , DNA Ribossômico/genética , Fezes/parasitologia , Helmintos/genética , Filogenia , RNA Ribossômico 18S/genética , Strongyloides stercoralis/patogenicidade , Estrongiloidíase/epidemiologia , Índias Ocidentais , Zoonoses/patologia
14.
Nat Commun ; 10(1): 2712, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221971

RESUMO

Clostridium difficile (C. difficile) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile. We demonstrate that IL-33 prevents C. difficile-associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile-associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.


Assuntos
Clostridium difficile/imunologia , Enterocolite Pseudomembranosa/imunologia , Imunidade Inata , Interleucina-33/metabolismo , Linfócitos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antibacterianos/efeitos adversos , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Clostridium difficile/patogenicidade , Colo/citologia , Colo/imunologia , Colo/microbiologia , Colo/patologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/mortalidade , Enterocolite Pseudomembranosa/terapia , Transplante de Microbiota Fecal , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Perfilação da Expressão Gênica , Humanos , Interleucina-33/imunologia , Linfócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Virulência/imunologia , Adulto Jovem
15.
Nat Microbiol ; 4(10): 1737-1749, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182797

RESUMO

Products derived from bacterial members of the gut microbiota evoke immune signalling pathways of the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. We recently demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities following depletion of bacteria, indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here, we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and interleukin (IL)-22-producing innate lymphoid cells, which in turn promote pSTAT3 signalling in intestinal epithelial cells and protection from intestinal injury. In addition, we demonstrate that MNV provides a striking IL-22-dependent protection against early-life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges.


Assuntos
Microbioma Gastrointestinal/imunologia , Interferon Tipo I/metabolismo , Interleucinas/metabolismo , Intestinos/imunologia , Intestinos/virologia , Animais , Antibacterianos/toxicidade , Proliferação de Células , Citrobacter rodentium/fisiologia , Colo/citologia , Colo/imunologia , Colo/metabolismo , Colo/virologia , Sulfato de Dextrana/toxicidade , Infecções por Enterobacteriaceae/prevenção & controle , Interleucinas/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Norovirus/imunologia , Norovirus/fisiologia , Transdução de Sinais/genética , Organismos Livres de Patógenos Específicos , Proteínas não Estruturais Virais/genética , Replicação Viral
16.
Colloids Surf B Biointerfaces ; 180: 212-220, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31054461

RESUMO

The objective of this study was to synthesize a novel antimicrobial cellulose nanofibril/silver nanoparticle (CNF/AgNP) nanocomposite by an ultraviolet (UV) irradiation method and evaluate the toxicity of the nanocomposite to human colon cells. AgNPs coated on CNFs have an average size of ˜28 nm and exhibited a surface plasma resonance absorption peak at 402 nm. Coating AgNPs on CNFs interfered with the formation of intra-chain and inter-chain hydrogen bonds of cellulose. Moreover, the CNF/AgNP nanocomposite exhibited significant antimicrobial activities against two important food-borne pathogens, including Escherichia coli O157:H7 and Staphylococcus aureus. No apparent toxicity of the CNF/AgNP nanocomposite to Caco-2 and FHC human colon cells was observed, except when a high content of (≥500 µg/m L) of the nanocomposite was used for which a significant decrease of cell viability was observed. The nanocomposite's toxicity was related to the content, size, and surface charge of UV-synthesized AgNPs on CNFs. These results indicate that the antimicrobial CNF/AgNP nanocomposite prepared by UV irradiation method can be potentially used as an active filler applied in food packaging materials.


Assuntos
Antibacterianos/farmacologia , Celulose/farmacologia , Nanopartículas Metálicas/toxicidade , Nanocompostos/toxicidade , Nanofibras/toxicidade , Prata/farmacologia , Testes de Toxicidade , Raios Ultravioleta , Células CACO-2 , Celulose/ultraestrutura , Colo/citologia , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/ultraestrutura , Humanos , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Nanocompostos/ultraestrutura , Nanofibras/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestrutura
17.
Microsc Res Tech ; 82(8): 1339-1344, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31070847

RESUMO

Inflammatory bowel disease (IBD) is a global, chronic intractable disease. The functions of drugs and food components have been evaluated in models of IBD induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Here, we used transmission (TEM) and osmium-maceration scanning (SEM) electron microscopy to evaluate the ultrastructure of colonic epithelial cells in rat models of IBD induced by TNBS. Histological evaluation revealed that the intestinal crypts in the most regions of the IBD-model colons were deformed and we classified them as having high cell migration rates (HMIG). The remaining regions in the intestinal crypts retained a relatively normal structure and we classified them as having low cell migration rates (LMIG). Osmium-maceration SEM revealed the mucosal fluid flowing in spaces without secretory granules in crypt goblet cells of both HMIG and LMIG regions, indicating the depletion of goblet cell mucin that is found in patients with IBD. The Golgi apparatus in absorptive cells was stacked and curled in both regions. Osmium-maceration SEM showed membrane network structures resembling endoplasmic reticulum that were large and expanded in absorptive cells with HMIG rather than with LMIG regions in IBD-model colons. These findings indicated that endoplasmic reticulum stress is associated with susceptibility to IBD and that the effects of various agents can be evaluated according to endoplasmic reticulum stress revealed by using electron microscopy in models of IBD induced by TNBS.


Assuntos
Colo/citologia , Células Epiteliais/ultraestrutura , Doenças Inflamatórias Intestinais/patologia , Animais , Colo/patologia , Modelos Animais de Doenças , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Células Epiteliais/patologia , Células Caliciformes/ultraestrutura , Complexo de Golgi/patologia , Complexo de Golgi/ultraestrutura , Doenças Inflamatórias Intestinais/induzido quimicamente , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mucinas , Ratos , Ácido Trinitrobenzenossulfônico/administração & dosagem
18.
Exp Parasitol ; 201: 34-41, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31034815

RESUMO

Ovine Eimeria spp. infections cause increased mortality, reduced welfare and substantial economic losses, and anticocccidials are important for their control. Recent reports of anticoccidial resistance against ovine Eimeria spp. necessitate the development of in vitro methods for the detection of reduced anticoccidial efficacy, especially since the in vivo methods are both expensive, time consuming and requires the use of otherwise healthy animals. The aim of the present study was therefore to approach a preliminary standardization of in vitro assays for evaluation of the efficacy of the most commonly used anticoccidials in ruminants. For this purpose, apart from the evaluation of inhibition of oocyst sporulation, most effort was concentrated on assessment of the capacity of the different anticoccidials to inhibit both the invasion and further development (up to the first schizogony) of E. ninakohlyakimovae sporozoites in bovine colonic epithelial cells (BCEC). For this purpose, infected cultures were monitored 1, 8 and 15 days post infection to determine the infection rate, number of immature schizonts and number, size and appearance of mature schizonts, respectively. No clear inhibitory effect was found with any of the anticoccidial formulations tested, and we could not identify why there were no measurable effects from the different anticoccidials. Despite the lack of positive results, further investigations should be encouraged, as this could decrease the need for animal experiments and could be used in the initial assessment of anticoccidial efficacy of new drugs.


Assuntos
Coccidiose/veterinária , Coccidiostáticos/farmacologia , Eimeria/efeitos dos fármacos , Doenças das Cabras/parasitologia , Animais , Bovinos , Células Cultivadas , Coccidiose/tratamento farmacológico , Coccidiose/parasitologia , Colo/citologia , Colo/parasitologia , Decoquinato/farmacologia , Resistência a Medicamentos , Eimeria/crescimento & desenvolvimento , Eimeria/isolamento & purificação , Células Epiteliais/parasitologia , Fezes/parasitologia , Doenças das Cabras/tratamento farmacológico , Cabras , Mucosa Intestinal/citologia , Mucosa Intestinal/parasitologia , Nitrilos/farmacologia , Oocistos/isolamento & purificação , Esquizontes/efeitos dos fármacos , Esquizontes/crescimento & desenvolvimento , Esporozoítos/isolamento & purificação , Sulfonamidas/farmacologia , Triazinas/farmacologia
19.
Eur J Pharmacol ; 853: 364-370, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31009637

RESUMO

Evidence suggests that propofol infusion syndrome (PRIS) is caused by an altered mitochondrial function. The aim of this study was to examine the effects of propofol and the vehicle MCT on mitochondrial function in hepatic and colonic tissue. Mitochondrial oxygen consumption was determined in colon and liver homogenates after incubation with buffer (control), propofol (50, 75, 100, 500  µM) or the carrier substances DMSO and MCT. State 2 (substrate-dependent) and state 3 (ADP-dependent respiration) were assessed. RCI (respiratory control index) - an indicator for coupling between electron transport chain system (ETS) and oxidative phosphorylation (OXPHOS) and ADP/O ratio - a parameter for efficacy of OXPHOS were calculated. Data were presented as % of control. In hepatic mitochondria, 500  µM propofol reduced RCI formulation-independently (propofol/MCT 500 µM: complex I: 66.3 ± 8.7%*, complex II: 75.5 ± 9.2%*; propofol/DMSO 500 µM: complex I: 29.1 ± 8.8%*, complex II: 49.3 ± 15.5%*). 75 â€¯µM Propofol/MCT reduced ADP/O for complex I (73.5 ± 27.3%*). DMSO did not affect hepatic mitochondria whereas MCT reduced RCI for complex II (87.2 ± 9.8%*) and ADP/O for complex I (93.7 ± 31.7%*). In colon 50 â€¯µM Propofol/MCT increased RCI for complex I and II (complex I: 127.2 ± 10.7%*, complex II: 136.8 ± 33.9%*) and 100 â€¯µM Propofol/MCT for complex I (131.4 ± 18.7%*). 500 â€¯µM Propofol/DMSO increased ADP/O for complex I (139.4 ± 41.4%*). DMSO did not affect RCI but increased ADP/O for both complexes (complex I: 119.9 ± 25.8%*, complex II: 110.2 ± 14.2%*). MCT increased RCI for complex I (123.0 ± 31.6%*). In hepatic mitochondria propofol uncoupled ETS from OXPHOS formulation-independently and propofol/MCT reduced efficacy of OXPHOS. In colonic mitochondria, propofol/MCT strengthened the coupling and propofol/DMSO enhanced the efficacy of OXPHOS.


Assuntos
Colo/citologia , Fígado/citologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Propofol/farmacologia , Animais , Respiração Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Especificidade de Órgãos , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar
20.
PLoS One ; 14(4): e0215122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30995271

RESUMO

BACKGROUND AND AIMS: The goal of the study was to assess calcium alone and Aquamin, a multi-mineral natural product that contains magnesium and detectable levels of 72 trace elements in addition to calcium, for capacity to affect growth and differentiation in colonoid cultures derived from histologically-normal human colon tissue. METHODS: Colonoid cultures were maintained in a low-calcium (0.25 mM) medium or in medium supplemented with an amount of calcium (1.5-3.0 mM), either from calcium alone or Aquamin for a period of two weeks. This was shown in a previous study to induce differentiation in colonoids derived from large adenomas. Changes in growth, morphological features and protein expression profile were assessed at the end of the incubation period using a combination of phase-contrast and scanning electron microscopy, histology and immunohistology, proteomic assessment and transmission electron microscopy. RESULTS: Unlike the previously-studied tumor-derived colonoids (which remained un-differentiated in the absence of calcium-supplementation), normal tissue colonoids underwent differentiation as indicated by gross and microscopic appearance, a low proliferative index and high-level expression of cytokeratin 20 in the absence of intervention (i.e., in control condition). Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). In spite of this, proteomic analysis and immunohistochemistry revealed that both interventions induced strong up-regulation of proteins that promote cell-cell and cell-matrix adhesive functions, barrier formation and tissue integrity. Transmission electron microscopy revealed an increase in desmosomes in response to intervention. CONCLUSIONS: These findings demonstrate that colonoids derived from histologically normal human tissue can undergo differentiation in the presence of a low ambient calcium concentration. However, higher calcium levels induce elaboration of proteins that promote cell-cell and cell-matrix adhesion. These changes could lead to improved barrier function and improved colon tissue health.


Assuntos
Adenoma/patologia , Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Junções Célula-Matriz/fisiologia , Colo/citologia , Adenoma/metabolismo , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colo/efeitos dos fármacos , Colo/metabolismo , Humanos , Minerais/farmacologia , Organoides/citologia , Organoides/metabolismo , Proteoma/análise
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