RESUMO
Inflammatory Bowel Disease (IBD) comprises a heterogeneous group of chronic inflammatory conditions of the gastrointestinal tract that include ulcerative colitis (UC) and Crohn's disease. Although the etiology is not well understood, IBD is characterized by a loss of the normal epithelium homeostasis that disrupts the intestinal barrier of these patients. Previous work by our group demonstrated that epithelial homeostasis along the colonic crypts involves a tight regulation of lipid profiles. To evaluate whether lipidomic profiles conveyed the functional alterations observed in the colonic epithelium of IBD, we performed matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) analyses of endoscopic biopsies from inflamed and non-inflamed segments obtained from UC patients. Our results indicated that lipid profiling of epithelial cells discriminated between healthy and UC patients. We also demonstrated that epithelial cells of the inflamed mucosa were characterized by a decrease in mono- and di-unsaturated fatty acid-containing phospholipids and higher levels of arachidonic acid-containing species, suggesting an alteration of the lipid gradients occurring concomitantly to the epithelial differentiation. This result was reinforced by the immunofluorescence analysis of EPHB2 and HPGD, markers of epithelial cell differentiation, sustaining that altered lipid profiles were at least partially due to a faulty differentiation process. Overall, our results showed that lipid profiling by MALDI-MSI faithfully conveys molecular and functional alterations associated with the inflamed epithelium, providing the foundation for a novel molecular characterization of UC patients.
Assuntos
Diferenciação Celular , Colo , Humanos , Colo/metabolismo , Colo/patologia , Masculino , Feminino , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Pessoa de Meia-Idade , Adulto , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Lipidômica/métodos , Enterócitos/metabolismo , Enterócitos/patologia , Metabolismo dos Lipídeos , Inflamação/metabolismo , Inflamação/patologia , Lipídeos/análise , Células Epiteliais/metabolismo , Células Epiteliais/patologiaRESUMO
Monogenetic variants are responsible for a range of congenital human diseases. Variants in genes that are important for intestinal epithelial function cause a group of disorders characterized by severe diarrhea and loss of nutrient absorption called congenital diarrheas and enteropathies (CODEs). CODE-causing genes include nutrient transporters, enzymes, structural proteins, and vesicular trafficking proteins in intestinal epithelial cells. Several severe CODE disorders result from the loss-of-function in key regulators of polarized endocytic trafficking such as the motor protein, Myosin VB (MYO5B), as well as STX3, STXBP2, and UNC45A. Investigations of the cell biology and pathophysiology following loss-of-function in these genes have led to an increased understanding of both homeostatic and pathological vesicular trafficking in intestinal epithelial cells. Modeling different CODEs through investigation of changes in patient tissues, coupled with the development of animal models and patient-derived enteroids, has provided critical insights into the enterocyte differentiation and function. Linking basic knowledge of cell biology with the phenotype of specific patient variants is a key step in developing effective treatments for rare monogenetic diseases. This knowledge can also be applied more broadly to our understanding of common epithelial disorders.
Assuntos
Enteropatias , Mucosa Intestinal , Animais , Humanos , Modelos Animais de Doenças , Enterócitos/metabolismo , Enterócitos/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Enteropatias/genética , Enteropatias/patologia , Enteropatias/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Modelos Biológicos , Diarreia/metabolismo , Diarreia/patologiaRESUMO
Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.
Assuntos
Gorduras na Dieta , Enterócitos , Metabolismo dos Lipídeos , Mitocôndrias , Animais , Camundongos , Aspartato-tRNA Ligase/metabolismo , Quilomícrons/metabolismo , Gorduras na Dieta/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Retículo Endoplasmático/metabolismo , Enterócitos/metabolismo , Enterócitos/patologia , Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Intestinos , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologiaRESUMO
Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Miosina Tipo V , Animais , Camundongos , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Enterócitos/metabolismo , Enterócitos/patologia , Genes Supressores de Tumor , Camundongos Knockout , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Miosinas , HumanosRESUMO
AIM: To evaluate the level of serum I-FABP (Fatty-Acid-Binding Protein a protein that binds fatty acids) and fecal zonulin as markers of the permeability of the mucous membrane of the small intestine in celiac patients. MATERIALS AND METHODS: A total of 151 celiac patients (25 men and 126 women) were examined. The median age was 42 years. Group I included 58 patients with newly diagnosed celiac disease; in group 2 38 patients, knowingly or unknowingly violating the gluten-free diet; group 3 consisted of 55 patients strictly observing gluten-free diet. The control group consisted of 20 healthy volunteers: 4 men and 16 women. All patients underwent esophagogastroduodenoscopy by biopsy of the mucous membrane of the small intestine and assessment of duodenobioptates according to Marsh. In the blood serum, the level of antibodies to tissue transglutaminase IgA and IgG was determined by the enzyme-linked immunosorbent assay using kits manufactured by Orgentec Diagnostics GmbH (Germany), the concentration of I-FABP in blood serum was determined using Hycult Biotech kits (Netherlands). The content of zonulin in feces was investigated by enzyme-linked immunosorbent assay using kits from Immundiagnostik AG (Germany). Statistical analysis was performed using the Statistica 13.3 software (StatSoft Inc., USA). RESULTS: There was a significant increase in the level of antibodies to tissue transglutaminase IgA [120.0 (41.1200)] IU/ml and IgG [31.4 (5.578.9)] IU/ml in patients of group 1 compared with group 2 [IgA 9.1 (2.987.6)] and IgG [3.8 (2.219.7)] IU/ml and group 3 [IgA 1.6 (1.03.2)] and IgG [2.2 (1.152.53)] (p0.01). The level of I-FABP in blood serum in patients of group 1 averaged 2045 pg/ml, in patients in group 2 1406 pg/ml, in patients in group 3 1000 pg/ml. All patients showed a significant increase in the mean I-FABP values compared to controls (1, 2 and control p0.01, 3 and control p=0.016). In patients with Marsh grade III AC atrophy, the I-FABP level depended on the degree of damage to the mucosa and significantly differed from the control: March IIIA (median: 1310 pg/ml, interquartile range: 12121461 pg/ml), March IIIB (median: 2090 pg/ml, interquartile range: 18122322 pg/ml) as well as Marsh IIIC (median: 2058 pg/ml, interquartile range 18582678 pg/ml). The concentration of zonulin in feces in patients of group 1 averaged 111.6 pg/mg, in patients of group 2 90.5 pg/mg. In patients of group 3 50 IU/ml. The concentration of zonulin in feces increased as the degree of mucosa atrophy increased (r=0.585, p0.01). However, despite the fact that both of these markers may indicate impaired permeability, each of them indicates damage to a certain level of the intestinal barrier, which is not always associated with the degree of mucosa atrophy. CONCLUSION: Determination of serum I-FABP and fecal zonulin levels in celiac patients allows for the assessment of intestinal permeability and can serve as non-invasive markers for monitoring ongoing structural changes in the mucosa without the need for endoscopy.
Assuntos
Doença Celíaca , Adulto , Feminino , Humanos , Masculino , Atrofia/metabolismo , Atrofia/patologia , Autoanticorpos , Biomarcadores , Doença Celíaca/diagnóstico , Enterócitos/patologia , Ácidos Graxos , Imunoglobulina A/metabolismo , Imunoglobulina G , Mucosa Intestinal/metabolismoRESUMO
Rationale: Serotonin (5-hydroxytryptamine, 5-HT) is generally considered to be involved in colitis-associated cancer (CAC), but previous research has yielded inconsistent results regarding the effect of 5-HT on CAC. 5-HT2B is one of the receptors of 5-HT, and the receptor is expressed in intestinal epithelial cells (IECs). However, the functions of 5-HT2B in CAC remain unclear. Our work demonstrates the variable functions of 5-HT/5-HT2B signaling in the initiation and progression of CAC in mice. Methods: We constructed two types of mutant mice homozygous knockout of Htr2b, the gene encoding 5-HT2B, in IECs (Htr2bΔIEC and Htr2bΔIEC-ER) to study the role of 5-HT2B in AOM/DSS-induced CAC model. Inflammation was measured using the body weight, colon length, and colitis severity score, and by histologic analysis of colon tissues. Tumor severity was assessed by tumor quantity, load, and histologic analysis of colon tumor tissues. Results: In Htr2bΔIEC mice, AOM/DSS induced an enhancement of colitis and tumor severity. This process was due to the inhibition of TGF-ß/SMAD signaling pathway and activation of IL-6/STAT3 signaling pathway. IL-6 antibody treatment reversed the stimulating effect of Htr2b deletion on tumorigenesis. However, tumor severity decreased in Htr2bΔIEC-ER mice injected with tamoxifen on day 48 of AOM/DSS treatment. Knockout Akt1 eliminated the function of 5-HT in promoting tumor cells. Conclusion: Our work elucidates 5-HT/5-HT2B/TGF-ß signaling as a critical tumor suppressing axis during CAC initiation but as a promoter of cancer progression in the late-stage of CAC. Our findings provide a new understanding of the role of 5-HT in the initiation and progression of CAC, offering a new perspective on the long-standing debate on whether the 5-HT signal promotes or inhibits tumors.
Assuntos
Neoplasias Associadas a Colite , Colite , Animais , Colite/induzido quimicamente , Sulfato de Dextrana , Enterócitos/metabolismo , Enterócitos/patologia , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Serotonina , Fator de Crescimento Transformador betaRESUMO
Iron is crucial to the regulation of the host innate immune system and the outcome of many infections. Hepatitis C virus (HCV), one of the major viral human pathogens that depends on iron to complete its life cycle, is highly skilled in evading the immune system. This study presents the construction and validation of a physiologically relevant triple-cell co-culture model that was used to investigate the input of iron in HCV infection and the interplay between HCV, iron, and determinants of host innate immunity. We recorded the expression patterns of key proteins of iron homeostasis involved in iron import, export and storage and examined their relation to the iron regulatory hormone hepcidin in hepatocytes, enterocytes and macrophages in the presence and absence of HCV. We then assessed the transcriptional profiles of pro-inflammatory cytokines Interleukin-6 (IL-6) and interleukin-15 (IL-15) and anti-inflammatory interleukin-10 (IL-10) under normal or iron-depleted conditions and determined how these were affected by infection. Our data suggest the presence of a link between iron homeostasis and innate immunity unfolding among liver, intestine, and macrophages, which could participate in the deregulation of innate immune responses observed in early HCV infection. Coupled with iron-assisted enhanced viral propagation, such a mechanism may be important for the establishment of viral persistence and the ensuing chronic liver disease.
Assuntos
Enterócitos/patologia , Hepatite C/patologia , Hepatócitos/patologia , Homeostase , Imunidade Inata , Ferro/metabolismo , Macrófagos/patologia , Técnicas de Cocultura , Citocinas/metabolismo , Enterócitos/imunologia , Enterócitos/metabolismo , Enterócitos/virologia , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepatite C/imunologia , Hepatite C/metabolismo , Hepatite C/virologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologiaRESUMO
Crohn's disease (CD) is an intestinal immune-dysfunctional disease. Extracellular vesicles (EVs) are membrane-enclosed particles full of functional molecules, e.g., nuclear acids. Recently, EVs have been shown to participate in the development of CD by realizing intercellular communication among intestinal cells. However, the role of EVs carrying double-strand DNA (dsDNA) shed from sites of intestinal inflammation in CD has not been investigated. Here we isolated EVs from the plasma or colon lavage of murine colitis and CD patients. The level of exosomal dsDNA, including mtDNA and nDNA, significantly increased in murine colitis and active human CD, and was positively correlated with the disease activity. Moreover, the activation of the STING pathway was verified in CD. EVs from the plasma of active human CD triggered STING activation in macrophages in vitro. EVs from LPS-damaged colon epithelial cells were also shown to raise inflammation in macrophages via activating the STING pathway, but the effect disappeared after the removal of exosomal dsDNA. These findings were further confirmed in STING-deficient mice and macrophages. STING deficiency significantly ameliorated colitis. Besides, potential therapeutic effects of GW4869, an inhibitor of EVs release were assessed. The application of GW4869 successfully ameliorated murine colitis by inhibiting STING activation. In conclusion, exosomal dsDNA was found to promote intestinal inflammation via activating the STING pathway in macrophages and act as a potential mechanistic biomarker and therapeutic target of CD.
Assuntos
Doença de Crohn/patologia , DNA/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Colite/patologia , Modelos Animais de Doenças , Endocitose/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Enterócitos/ultraestrutura , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/ultraestrutura , Humanos , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Proteínas de Membrana/deficiência , Camundongos Knockout , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The cellular and tissue damage induced by oxidative stress (OS) contribute to a variety of human diseases, which include gastrointestinal diseases. Salvianolic acid B (Sal B), which is a natural polyphenolic acid in Salvia miltiorrhiza, exhibits prominent antioxidant properties. However, its precise function and molecular mechanisms in protecting normal intestine epithelium from OS-induced damage are still poorly defined. In this study, we tried to clarify this relationship. Here, we found Sal B addiction in the rat intestinal epithelial cell, IEC-6, prevented H2O2-induced cell viability decrease and apoptosis induction, ameliorated H2O2-induced intestinal epithelial barrier dysfunction and mitochondrial dysfunction, and suppressed H2O2-induced production of ROS to varying degrees, ranging from 10% to 30%. Moreover, by employing an ischemia reperfusion model of rats, we also discovered that Sal B treatment reversed ischemia and a reperfusion-caused decrease in villus height and crypt depth, decreased proliferation of enterocytes, and increased the apoptotic index in the jejunum and ileum. Mechanistically, Sal B treatment up-regulated the phosphorylated level of Akt and GSK3ß in enterocytes in vitro and in vivo, and PI3K inhibitor LY294002 treatment abrogated the protective effects of Sal B. Meanwhile, the inactivation of GSK3ß reversed the oxidative stress-induced apoptosis and mitochondrial dysfunction in IEC-6 cells. Together, our results demonstrated that the damage of intestinal epithelial cells in in vitro and in vivo models were both attenuated by Sal B treatment, and such antioxidant activity might very possibly be attributed to the activation of Akt/GSK3ß signaling.
Assuntos
Antioxidantes/farmacologia , Benzofuranos/farmacologia , Enteropatias/tratamento farmacológico , Animais , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Benzofuranos/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Íleo/citologia , Íleo/efeitos dos fármacos , Íleo/patologia , Enteropatias/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Jejuno/citologia , Jejuno/efeitos dos fármacos , Jejuno/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Salvia miltiorrhiza/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Tissue homeostasis is achieved by balancing stem cell maintenance, cell proliferation and differentiation, as well as the purging of damaged cells. Elimination of unfit cells maintains tissue health; however, the underlying mechanisms driving competitive growth when homeostasis fails, for example, during tumorigenesis, remain largely unresolved. Here, using a Drosophila intestinal model, we find that tumor cells outcompete nearby enterocytes (ECs) by influencing cell adhesion and contractility. This process relies on activating the immune-responsive Relish/NF-κB pathway to induce EC delamination and requires a JNK-dependent transcriptional upregulation of the peptidoglycan recognition protein PGRP-LA. Consequently, in organisms with impaired PGRP-LA function, tumor growth is delayed and lifespan extended. Our study identifies a non-cell-autonomous role for a JNK/PGRP-LA/Relish signaling axis in mediating death of neighboring normal cells to facilitate tumor growth. We propose that intestinal tumors "hijack" innate immune signaling to eliminate enterocytes in order to support their own growth.
Assuntos
Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Imunidade Inata/genética , MAP Quinase Quinase 4/genética , Neoplasias/genética , Fatores de Transcrição/genética , Animais , Adesão Celular/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Enterócitos/metabolismo , Enterócitos/patologia , Humanos , Intestinos/crescimento & desenvolvimento , Intestinos/patologia , Mecanotransdução Celular/genética , NF-kappa B/genética , Neoplasias/patologia , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Intrinsic autophagy is important for the maintenance of intestinal homeostasis and intestinal regeneration. Ionizing radiation suppresses intrinsic autophagy and reduces damage-induced regeneration in the intestine, resulting in intestinal injury. Resveratrol, a sirtuin 1 (SIRT1) agonist, promotes autophagy and exerts radioprotective effect. In this study, the protective effect of resveratrol against radiation-induced intestinal injury and its potential mechanism were investigated. Intestinal epithelial cells (IEC-6) were exposed to 10 Gy ionizing radiation and resveratrol (0.1-40.0 µM). Cell viability was investigated using Cell Counting Kit 8 (CCK8), apoptosis was observed by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry, and the expression of apoptotic and autophagic proteins was determined by western blotting. Resveratrol exerted a high toxicity against IEC-6 cells, but at low concentrations, it inhibited ionizing radiation-induced apoptosis. Resveratrol increased SIRT1 expression after irradiation and inhibited ionizing radiation-induced p53 acetylation and pro-apoptotic protein, Bax, expression. Furthermore, resveratrol promoted autophagy via the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, thereby protecting IEC-6 cells against radiation-induced damage. These results suggest that resveratrol reduces radiation-induced IEC-6 cell damage by inhibiting apoptosis and promoting autophagy via the activation of SIRT1, and that the PI3K/AKT/mTOR signaling pathway is involved in the induction of autophagy.
Assuntos
Apoptose , Autofagia , Citoproteção , Enterócitos/patologia , Radiação Ionizante , Resveratrol/farmacologia , Sirtuína 1/metabolismo , Acetilação/efeitos dos fármacos , Acetilação/efeitos da radiação , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Autofagia/efeitos dos fármacos , Autofagia/efeitos da radiação , Proteína Beclina-1/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Citoproteção/efeitos dos fármacos , Citoproteção/efeitos da radiação , Enterócitos/efeitos dos fármacos , Enterócitos/efeitos da radiação , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Organoids allow the recapitulation of intestinal homeostasis and cancerogenesis in vitro; however, RNA sequencing (RNA-seq)-based methods for drug screens are missing. We develop targeted organoid sequencing (TORNADO-seq), a high-throughput, high-content drug discovery platform that uses targeted RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in organoids. TORNADO-seq is a fast, highly reproducible time- and cost-effective ($5 per sample) method that can probe cell mixtures and their differentiation state in the intestinal system. We apply this method to isolate drugs that enrich for differentiated cell phenotypes and show that these drugs are highly efficacious against cancer compared to wild-type organoids. Furthermore, TORNADO-seq facilitates in-depth insight into the mode of action of these drugs. Our technology can easily be adapted to many other systems and will allow for more systematic, large-scale, and quantitative approaches to study the biology of complex cellular systems.
Assuntos
Antineoplásicos/farmacologia , Detecção Precoce de Câncer/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Organoides/efeitos dos fármacos , Medicamentos sob Prescrição/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos/química , Antineoplásicos/classificação , Diferenciação Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/patologia , Redes Reguladoras de Genes , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Ensaios de Triagem em Larga Escala , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Organoides/metabolismo , Organoides/patologia , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Medicamentos sob Prescrição/química , Medicamentos sob Prescrição/classificação , RNA-Seq , Análise de Sequência de RNA , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/classificaçãoRESUMO
Adenocarcinoma with enteroblastic differentiation is an extremely rare tumor with poor prognosis and unique pathologic features. The tumor appears to be relatively more common in stomach, with rare cases reported in esophagus, colon, rectum and ampulla. Underrecognition by pathologists may be a contributing factor towards underreporting of this tumor. Combination of carcinosarcoma and enteroblastic differentiation has not been reported so far.We report a unique case of ampullary carcinosarcoma with enteroblastic differentiation in a 59-year-old female, diagnosed in the pancreatoduodenectomy specimen. The carcinomatous component showed features of enteroblastic differentiation characterized by tubular architecture with clear cytoplasm, solid component with trabecular architecture and immunohistochemical expression of SALL4 and AFP. The patient was treated with adjuvant Folfirinox chemotherapy and is disease free at 17 months follow up.
Assuntos
Carcinossarcoma/diagnóstico , Carcinossarcoma/fisiopatologia , Mucosa Intestinal/citologia , Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/genética , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Pancreaticoduodenectomia/métodos , Neoplasias Gástricas/patologiaRESUMO
Rho guanine nucleotide exchange factors (RhoGEFs) regulate Rho GTPase activity and cytoskeletal and cell adhesion dynamics. ßPix, a CDC42/RAC family RhoGEF encoded by ARHGEF7, is reported to modulate human colon cancer cell proliferation and postwounding restitution of rat intestinal epithelial monolayers. We hypothesized that ßPix plays a role in maintaining intestinal epithelial homeostasis. To test this hypothesis, we examined ßPix distribution in the human and murine intestine and created mice with intestinal epithelial-selective ßPix deletion [ßPixflox/flox/Tg(villin-Cre); Arhgef7 CKO mice]. Using Arhgef7 conditional knockout (CKO) and control mice, we investigated the consequences of ßPix deficiency in vivo on intestinal epithelial and enteroid development, dextran sodium sulfate-induced mucosal injury, and gut permeability. In normal human and murine intestines, we observed diffuse cytoplasmic and moderate nuclear ßPix immunostaining in enterocytes. Arhgef7 CKO mice were viable and fertile, with normal gross intestinal architecture but reduced small intestinal villus height, villus-to-crypt ratio, and goblet cells; small intestinal crypt cells had reduced Ki67 staining, compatible with impaired cell proliferation. Enteroids derived from control mouse small intestine were viable for more than 20 passages, but those from Arhgef7 CKO mice did not survive beyond 24 h despite addition of Wnt proteins or conditioned media from normal enteroids. Adding a Rho kinase (ROCK) inhibitor partially rescued CKO enteroid development. Compared with littermate control mice, dextran sodium sulfate-treated ßPix-deficient mice lost more weight and had greater impairment of intestinal barrier function, and more severe colonic mucosal injury. These findings reveal ßPix expression is important for enterocyte development, intestinal homeostasis, and resistance to toxic injury.NEW & NOTEWORTHY To explore the role of ßPix, a guanine nucleotide exchange factor encoded by ARHGEF7, in intestinal development and physiology, we created mice with intestinal epithelial cell Arhgef7/ßPix deficiency. We found ßPix essential for normal small intestinal epithelial cell proliferation, villus development, and mucosal resistance to injury. Moreover, Rho kinase signaling mediated developmental arrest observed in enteroids derived from ßPix-deficient small intestinal crypts. Our studies provide insights into the role Arhgef7/ßPix plays in intestinal epithelial homeostasis.
Assuntos
Proliferação de Células , Colite/metabolismo , Colo/metabolismo , Enterócitos/metabolismo , Mucosa Intestinal/metabolismo , Microvilosidades/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/deficiência , Animais , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Enterócitos/patologia , Feminino , Deleção de Genes , Humanos , Mucosa Intestinal/patologia , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvilosidades/patologia , Organoides , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos , Quinases Associadas a rho/metabolismoRESUMO
Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.
Assuntos
Neoplasias do Colo/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Imunofluorescência , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Coloração e RotulagemRESUMO
We report a probable case of abetalipoproteinemia in an infant who presented with unusual symptoms of late-onset vitamin K deficiency. Abetalipoproteinemia is a rare autosomal recessive disease caused by mutation of the microsomal triglyceride transfer protein gene, resulting in the absence of microsomal triglyceride transfer protein function in the small bowel. It is characterized by the absence of plasma apolipoprotein B-containing lipoproteins, fat malabsorption, hypocholesterolemia, retinitis pigmentosa, progressive neuropathy, myopathy, and acanthocytosis. A biopsy of the small intestine characteristically shows marked lipid accumulation in the villi of enterocytes. Large supplements of fat-soluble vitamins A, D, E, and K have been shown to limit neurologic and ocular manifestations. Dietary fat intake is limited to medium-chain triglycerides.
Assuntos
Abetalipoproteinemia/complicações , Deficiência de Vitamina K/complicações , Abetalipoproteinemia/sangue , Abetalipoproteinemia/diagnóstico , Abetalipoproteinemia/patologia , Duodeno/patologia , Enterócitos/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Deficiência de Vitamina K/sangue , Deficiência de Vitamina K/diagnóstico , Deficiência de Vitamina K/patologiaRESUMO
BACKGROUND AND AIMS: Cannabinoid receptor [CB] activation can attenuate inflammatory bowel disease [IBD] in experimental models and human cohorts. However, the roles of the microbiome, metabolome, and the respective contributions of haematopoietic and non-haematopoietic cells in the anti-colitic effects of cannabinoids have yet to be determined. METHODS: Female C57BL/6 mice were treated with either cannabidiol [CBD], Δâ9-tetrahydrocannabinol [THC], a combination of CBD and THC, or vehicle, in several models of chemically induced colitis. Clinical parameters of colitis were assessed by colonoscopy, histology, flow cytometry, and detection of serum biomarkers; single-cell RNA sequencing and qRT-PCR were used to evaluate the effects of cannabinoids on enterocytes. Immune cell transfer from CB2 knockout mice was used to evaluate the contribution of haematopoietic and non-haematopoietic cells to colitis protection. RESULTS: We found that THC prevented colitis and that CBD, at the dose tested, provided little benefit to the amelioration of colitis, nor when added synergistically with THC. THC increased colonic barrier integrity by stimulating mucus and tight junction and antimicrobial peptide production, and these effects were specific to the large intestine. THC increased colonic Gram-negative bacteria, but the anti-colitic effects of THC were independent of the microbiome. THC acted both on immune cells via CB2 and on enterocytes, to attenuate colitis. CONCLUSIONS: Our findings demonstrate how cannabinoid receptor activation on both immune cells and colonocytes is critical to prevent colonic inflammation. These studies also suggest how cannabinoid receptor activation can be used as a preventive and therapeutic modality against colitis.
Assuntos
Canabidiol/farmacologia , Colite , Dronabinol/farmacologia , Enterócitos , Imunidade Celular , Receptor CB2 de Canabinoide , Animais , Agonistas de Receptores de Canabinoides/farmacologia , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Colite/terapia , Colonoscopia/métodos , Monitoramento de Medicamentos , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Substâncias Protetoras/farmacologia , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismoRESUMO
Occurrence of Colorectal cancer (CRC) is relevant with gut microbiota. However, role of IRF3, a key signaling mediator in innate immune sensing, has been barely investigated in CRC. Here, we unexpectedly found that the IRF3 deficient mice are hyper-susceptible to the development of intestinal tumor in AOM/DSS and Apcmin/+ models. Genetic ablation of IRF3 profoundly promotes the proliferation of intestinal epithelial cells via aberrantly activating Wnt signaling. Mechanically, IRF3 in resting state robustly associates with the active ß-catenin in the cytoplasm, thus preventing its nuclear translocation and cell proliferation, which can be relieved upon microbe-induced activation of IRF3. In accordance, the survival of CRC is clinically correlated with the expression level of IRF3. Therefore, our study identifies IRF3 as a negative regulator of the Wnt/ß-catenin pathway and a potential prognosis marker for Wnt-related tumorigenesis, and describes an intriguing link between gut microbiota and CRC via the IRF3-ß-catenin axis.
Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Núcleo Celular/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/prevenção & controle , Fator Regulador 3 de Interferon/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Enterócitos/metabolismo , Enterócitos/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Antígeno Ki-67/metabolismo , Camundongos Endogâmicos C57BL , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Análise de Sobrevida , Via de Sinalização Wnt , beta Catenina/químicaRESUMO
Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.
Assuntos
Doenças dos Peixes/microbiologia , Microsporídios/isolamento & purificação , Microsporidiose/veterinária , Dourada/microbiologia , Animais , Aquicultura , Núcleo Celular/microbiologia , Núcleo Celular/patologia , Citoplasma/microbiologia , Citoplasma/patologia , Enterócitos/microbiologia , Enterócitos/patologia , Doenças dos Peixes/patologia , Granulócitos/microbiologia , Granulócitos/patologia , Granuloma/microbiologia , Granuloma/patologia , Histocitoquímica/veterinária , Imunidade Celular , Hibridização In Situ/veterinária , Intestinos/microbiologia , Intestinos/patologia , Microsporídios/classificação , Microsporídios/ultraestrutura , Microsporidiose/patologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Dourada/crescimento & desenvolvimentoRESUMO
The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells, through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu+ revival stem cells (revSCs). However, the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes, that are hyperproliferative, yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0-/- enterocytes show basal induction of the Clu+ regenerative program and a fetal gene expression signature marked by Sca1, but upon injury are unable to generate Sca-1+/Clu+ revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity.