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The use of the anti-inflammatory drug indomethacin (INDO) in preterm infants has been associated with an increased risk of developing enteropathies. In this study, we have investigated the direct impact of INDO on the human mid-gestation intestinal transcriptome using serum-free organ culture. After determining the optimal dose of 1 µM of INDO (90% inhibition of intestinal prostaglandin E2 production and range of circulating levels in treated preterm babies), global gene expression profiles were determined using Illumina bead chip microarrays in both small and large intestines after 48 h of INDO treatment. Using Ingenuity Pathway Analysis software, we identified critical metabolic pathways that were significantly altered by INDO in both intestinal segments including inflammation and also glycolysis, oxidative phosphorylation and free radical scavenging/oxidoreductase activity, which were confirmed by qPCR at the level of individual genes. Taken together, these data revealed that INDO directly exerts multiple detrimental effects on the immature human intestine.
Assuntos
Indometacina/administração & dosagem , Redes e Vias Metabólicas/genética , Nascimento Prematuro/metabolismo , Transcriptoma/efeitos dos fármacos , Feto Abortado/metabolismo , Feto Abortado/patologia , Anti-Inflamatórios/administração & dosagem , Feminino , Humanos , Mucosa Intestinal/metabolismo , Intestinos/fisiopatologia , Técnicas de Cultura de Órgãos , Gravidez , Nascimento Prematuro/tratamento farmacológico , Nascimento Prematuro/fisiopatologia , Transcriptoma/genéticaRESUMO
In this paper, deep learning-based techniques for film grain removal and synthesis that can be applied in video coding are proposed. Film grain is inherent in analog film content because of the physical process of capturing images and video on film. It can also be present in digital content where it is purposely added to reflect the era of analog film and to evoke certain emotions in the viewer or enhance the perceived quality. In the context of video coding, the random nature of film grain makes it both difficult to preserve and very expensive to compress. To better preserve it while compressing the content efficiently, film grain is removed and modeled before video encoding and then restored after video decoding. In this paper, a film grain removal model based on an encoder-decoder architecture and a film grain synthesis model based on a conditional generative adversarial network (cGAN) are proposed. Both models are trained on a large dataset of pairs of clean (grain-free) and grainy images. Quantitative and qualitative evaluations of the developed solutions were conducted and showed that the proposed film grain removal model is effective in filtering film grain at different intensity levels using two configurations: 1) a non-blind configuration where the film grain level of the grainy input is known and provided as input; and 2) a blind configuration where the film grain level is unknown. As for the film grain synthesis task, the experimental results show that the proposed model is able to reproduce realistic film grain with a controllable intensity level specified as input.
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The inflammatory response of the preterm infants' intestine underlines its inability to respond to hemodynamic stress, microbes, and nutrients. Recent evidence suggests that exogenous epidermal growth factor (EGF) exerts a therapeutic influence on neonatal enteropathies. However, the molecular mechanisms underlying the beneficial effects of EGF remain to be clarified. The purpose of this study was to evaluate the impact of EGF on the gene expression profiles of the developing human small and large intestine at midgestation in serum-free organ cultures using microarrays. The gene expression profiles of cultured human fetal ileal and colonic explants were investigated in the absence or presence of a physiological concentration of 50 ng/ml EGF for 48 h. Data were analyzed with the Ingenuity Pathway Analysis (IPA) software and confirmed by qPCR. We found a total of 6,474 differentially expressed genes in the two segments in response to EGF. IPA functional analysis revealed that in addition to differentially modulating distinct cellular, molecular, and physiological functions in the small and large intestine, EGF regulated the inflammatory response in both intestinal segments in a distinct manner. For instance, several intestinal-derived chemokines such as CCL2, CCL25, CXCL5, and CXCL10 were found to be differentially regulated by EGF in the immature ileum and colon. The findings showing the anti-inflammatory influence of exogenous EGF suggests a mechanistic basis for the beneficial effects of EGF on neonatal enteropathies. These results reinforce growing evidence that by midgestation, the human small intestine and colon rely on specific and distinct regulatory pathways.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL5/metabolismo , Quimiocinas CC/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Humanos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Intestinos/embriologia , Técnicas de Cultura de ÓrgãosRESUMO
Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor mainly expressed in the liver, intestine, kidney, and pancreas. Many of its hepatic and pancreatic functions have been described, but limited information is available on its role in the gastrointestinal tract. The objectives of this study were to evaluate the anti-inflammatory and antioxidant functions of HNF4α as well as its implication in intestinal lipid transport and metabolism. To this end, the HNF4A gene was knocked down by transfecting Caco-2 cells with a pGFP-V-RS lentiviral vector containing an shRNA against HNF4α. Inactivation of HNF4α in Caco-2 cells resulted in the following: (a) an increase in oxidative stress as demonstrated by the levels of malondialdehyde and conjugated dienes; (b) a reduction in secondary endogenous antioxidants (catalase, glutathione peroxidase, and heme oxygenase-1); (c) a lower protein expression of nuclear factor erythroid 2-related factor that controls the antioxidant response elements-regulated antioxidant enzymes; (d) an accentuation of cellular inflammatory activation as shown by levels of nuclear factor-κB, interleukin-6, interleukin-8, and leukotriene B4; (e) a decrease in the output of high density lipoproteins and of their anti-inflammatory and anti-oxidative components apolipoproteins (apo) A-I and A-IV; (f) a diminution in cellular lipid transport revealed by a lower cellular secretion of chylomicrons and their apoB-48 moiety; and (g) alterations in the transcription factors sterol regulatory element-binding protein 2, peroxisome proliferator-activated receptor α, and liver X receptor α and ß. In conclusion, HNF4α appears to play a key role in intestinal lipid metabolism as well as intestinal anti-oxidative and anti-inflammatory defense mechanisms.
Assuntos
Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipoproteínas/biossíntese , Estresse Oxidativo/fisiologia , Antioxidantes/metabolismo , Transporte Biológico/fisiologia , Células CACO-2 , Células Epiteliais/citologia , Técnicas de Silenciamento de Genes , Fator 4 Nuclear de Hepatócito , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-6/biossíntese , Interleucina-6/genética , Interleucina-8/biossíntese , Interleucina-8/genética , Mucosa Intestinal/citologia , Leucotrieno B4/biossíntese , Leucotrieno B4/genética , Lipoproteínas/genética , Receptores X do Fígado , Malondialdeído/metabolismo , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
Helicobacter pylori infects more than half of the human population worldwide. In the absence of treatment, this persistent infection leads to asymptomatic gastritis, which in some cases can progress into gastric ulcers and adenocarcinomas. The host-microbial interactions that govern the clinical outcome of infection remain incompletely understood. H. pylori is known to disrupt gastric epithelial tight junctions, which may represent a significant component of disease pathogenesis. The present study demonstrates that H. pylori disrupt epithelial tight junctional claudin-4 in a Rho kinase (ROCK)-dependent manner in human gastric epithelial (HGE-20) cell monolayers, independently of the virulence factors CagA and VacA, and without altering claudin-4 transcription. In the same epithelial cell model, interleukin (IL)-1beta, mediated a similar ROCK-dependent pattern of tight junction disruption. Further experiments revealed that H. pylori infection induced IL-1 receptor type I (IL-1RI) phosphorylation, independently of epithelial secretion of its endogenous ligands IL-1alpha, IL-1beta or IL-18. Finally, inhibition of IL-1RI activation prevented H. pylori-induced ROCK activation and claudin-4 disruption. Taken together, these findings identify a novel pathophysiological mechanism by which H. pylori disrupts gastric epithelial barrier structure via IL-1RI-dependent activation of ROCK, which in turn mediates tight junctional claudin-4 disruption.
Assuntos
Células Epiteliais/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Proteínas de Membrana/antagonistas & inibidores , Receptores de Interleucina-1/metabolismo , Quinases Associadas a rho/metabolismo , Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Linhagem Celular , Claudina-4 , Humanos , Interleucina-1beta/metabolismo , Fosforilação , Fatores de Virulência/fisiologiaRESUMO
BACKGROUND AND OBJECTIVES: The occurrence of many neonatal inflammatory intestinal diseases in preterm infants highlights the susceptibility of the immature intestine to responding inadequately to nutrients and microbes. A better understanding of functional intestinal development is essential for the design of optimal treatments ensuring survival and growth of premature infants. The purpose of this study was to evaluate the gene expression profiles of the human ileum and colon at mid-gestation because these 2 segments are considered to be similar at this stage and are the sites of the most frequent pathologies in preterm infants. SUBJECTS AND METHODS: We compared the gene-expression profiles of human fetal small and large intestines using a cDNA microarray and analyzed the data with Ingenuity Pathway Analysis software. RESULTS: We found that a significant proportion of the genes was differentially expressed in the 2 segments. Gene cluster analysis revealed an even higher level of transcriptional dissimilarity at the functional level. For instance, segment-specific/overexpressed gene clusters in the ileum included genes involved with amino acid, vitamin, and mineral metabolism, reflecting the higher level of maturity of the small intestine as compared with the colon in which genes involved with cell cycle, cell death, and cell signaling were the predominant clusters of genes expressed. CONCLUSIONS: Functional clustering analysis of the differentially expressed genes revealed important functional differences between the 2 segments and a relative immaturity of the colon, suggesting that already at mid-gestation, the 2 intestinal segments should be considered as 2 distinct organs.
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Colo/embriologia , Expressão Gênica/fisiologia , Íleo/embriologia , Ciclo Celular/genética , Morte Celular/genética , Análise por Conglomerados , DNA Complementar , Perfilação da Expressão Gênica/métodos , Humanos , Análise em Microsséries , Transdução de Sinais/genéticaRESUMO
The integrin alpha6 subunit exists as two different variants, termed alpha6A and alpha6B. These two variants have been shown to harbor potentially distinct biochemical properties but little is known about their cellular function. The aim of this work was to characterize the expression of the integrin alpha6A and B variants in relation to cell proliferation and differentiation in the human small intestinal epithelium. The results showed distinct expression patterns for the two variants along the crypt-villus axis. Indeed, proliferative cells of the crypt were found to predominantly express alpha6A, while differentiated enterocytes and Paneth cells expressed the alpha6B variant. A similar relationship was observed in intestinal cell models by competitive RT-PCR. Further studies in the Caco-2 cell model showed that manipulating the cellular balance of the two alpha6 variants can influence transcriptional activities related to cell proliferation but not differentiation. This suggests that differential expression of the alpha6 subunits is involved in the intestinal epithelial cell renewal process. Further studies will be needed to substantiate this hypothesis.
Assuntos
Diferenciação Celular/fisiologia , Integrina alfa6/metabolismo , Integrina beta4/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Células CACO-2 , Proliferação de Células , Humanos , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Isoformas de Proteínas/metabolismoRESUMO
Although intestinal (I) and liver (L) fatty acid binding proteins (FABP) have been widely studied, the physiological significance of the presence of the two FABP forms (I- and L-FABP) in absorptive cells remains unknown as do the differences related to their distribution along the crypt-villus axis, regional expression, ontogeny and regulation in the human intestine. Our morphological experiments supported the expression of I- and L-FABP as early as 13 weeks of gestation. Whereas cytoplasmic immunofluorescence staining of L-FABP was barely detectable in the lower half of the villus and in the crypt epithelial cells, I-FABP was visualized in epithelial cells of the crypt-villus axis in all intestinal segments until the adult period in which the staining was maximized in the upper part of the villus. Immunoelectron microscopy revealed more intense labeling of L-FABP compared with I-FABP, accompanied with a heterogeneous distribution in the cytoplasm, microvilli and basolateral membranes. By western blot analysis, I- and L-FABP at 15 weeks of gestation appeared predominant in jejunum compared with duodenum, ileum, proximal and distal colon. Exploration of the maturation aspect documented a rise in L-FABP in adult tissues. Permanent transfections of Caco-2 cells with I-FABP cDNA resulted in decreased lipid export, apolipoprotein (apo) biogenesis and chylomicron secretion. Additionally, supplementation of Caco-2 with insulin, hydrocortisone and epidermal growth factor differentially modulated the expression of I- and L-FABP, apo B-48 and microsomal triglyceride transfer protein (MTP), emphasizing that these key proteins do not exhibit a parallel modulation. Overall, our findings indicate that the two FABPs display differences in localization, regulation and developmental pattern.
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Colo/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Jejuno/metabolismo , Lipoproteínas/metabolismo , Células CACO-2 , Colo/embriologia , Colo/crescimento & desenvolvimento , Humanos , Lactente , Jejuno/embriologia , Jejuno/crescimento & desenvolvimento , Especificidade de ÓrgãosRESUMO
The morphological development of the gastrointestinal tract (GI), in laboratory animals as well as in humans, has been well described since more than 100 years. However, even though its functional development and regulatory mechanisms are pretty well understood, our knowledge of the human GI functions originated primarily from studies on rat and mouse. Because of clear differences in genetic make up, development rates and sequences, as well as physiological differences, extrapolations of animal data to the human must be made with caution. A reliable organ culture technique in which the morphological as well as physiological parameters are well maintained has been set up. This technique allows studies of basic physiological functions such as gene expression, localization of specific cell markers, numerous digestive enzymatic activities, and lipid and lipoprotein processing. Furthermore, it also permits to determine and characterize the biological actions of potential regulators such as growth factors and hormones. Finally, the establishment of human intestinal epithelial cell lines allows the validation and the characterization of the molecular mechanisms involved in the specific regulatory pathways of the human GI development.
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Trato Gastrointestinal/embriologia , Apolipoproteínas/biossíntese , Western Blotting , Células CACO-2 , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Trato Gastrointestinal/metabolismo , Humanos , Imunoprecipitação , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Block partition structure is a critical module in video coding scheme to achieve significant gap of compression performance. Under the exploration of the future video coding standard, named Versatile Video Coding (VVC), a new Quad Tree Binary Tree (QTBT) block partition structure has been introduced. In addition to the QT block partitioning defined in High Efficiency Video Coding (HEVC) standard, new horizontal and vertical BT partitions are enabled, which drastically increases the encoding time compared to HEVC. In this paper, we propose a lightweight and tunable QTBT partitioning scheme based on a Machine Learning (ML) approach. The proposed solution uses Random Forest classifiers to determine for each coding block the most probable partition modes. To minimize the encoding loss induced by misclassification, risk intervals for classifier decisions are introduced in the proposed solution. By varying the size of risk intervals, tunable trade-off between encoding complexity reduction and coding loss is achieved. The proposed solution implemented in the JEM-7.0 software offers encoding complexity reductions ranging from 30average for only 0.7% to 3.0% Bjxntegaard Delta Rate (BDBR) increase in Random Access (RA) coding configuration, with very slight overhead induced by Random Forest. The proposed solution based on Random Forest classifiers is also efficient to reduce the complexity of the Multi-Type Tree (MTT) partitioning scheme under the VTM-5.0 software, with complexity reductions ranging from 25% to 61% in average for only 0.4% to 2.2% BD-BR increase.
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Restitution is a crucial event during the healing of superficial injury of the gastric mucosa involving epithelial cell sheet movement into the damaged area. We demonstrated that growth factors promote the restitution of human gastric epithelial cells. However, the intracellular signaling pathways that transmit extracellular cues as well as regulate basal and growth factor-stimulated gastric epithelial cell migration are still unclear. Herein, confluent human gastric epithelial cell monolayers (HGE-17) or primary cultures of gastric epithelial cells were wounded with a razor blade and the migration response was analyzed in presence or absence of TGFalpha or of pharmacological inhibitors of signaling proteins. Kinase activation profile analysis and phase-contrast microscopy were also performed in parallel. We report that ERK1/2 and Akt activities are rapidly stimulated following wounding of HGE-17 cells. Treatment of confluent HGE-17 cells or primary cultures of gastric epithelial cells with the phosphatidylinositol 3-kinase inhibitor LY294002, but not the MEK1 inhibitor, PD98059, significantly inhibits basal and TGFalpha-induced migration following wounding. Conversely, treatment of wounded HGE-17 cells with phosphatidylinositol(3,4,5)-triphosphate is sufficient to stimulate basal cell migration by 235%. In addition, pp60c-src kinase activity and tyrosine phosphorylation of epidermal growth factor receptors (EGFR) are also rapidly enhanced after wounding and pharmacological inhibition of both these activities strongly attenuates basal and TGFalpha-induced migration as well as Akt phosphorylation levels. In conclusion, the present results indicate that EGFR-dependent PI3K activation promotes restitution of wounded human gastric epithelial monolayers.
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Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Estômago/citologia , Cicatrização , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Morfolinas/farmacologia , Fosfatos de Fosfatidilinositol/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Fator de Crescimento Transformador alfa/farmacologia , Tirosina/metabolismoRESUMO
Mechanisms that specifically modulate cell spreading and/or cell migration following epithelial wounding are poorly understood. Using micro-wounded human gastric epithelial monolayers, we show herein that EGF and TGFalpha maximally increase spreading of epithelial sheets under a cell proliferation-independent mechanism. Treatment of confluent HGE-17 cells with the phosphatidylinositol 3-kinase inhibitor, LY294002, and the epidermal growth factor receptor inhibitor, PD153035, strongly reduced basal and TGFalpha-stimulated cell spreading. While pharmacological inhibition of pp60src-kinase activity also attenuated basal epithelial spreading, addition of the mTOR/p70S6K inhibitor rapamycin or a specific siRNA targeting ILK sequence did not affect the kinetic rates of wound closure. Epithelial wound healing was initiated by actin purse-string contraction followed by lamellae formation. Conversely, disruption of actin and tubulin stability with cytochalasin D and nocodazole, respectively, inhibited epithelial sheet spreading. Finally, antibodies directed against the alpha3 integrin subunit, but not against the alpha6 or alpha2 subunits, attenuated epithelial sheet spreading as well as lamellae formation. In conclusion, the current investigation establishes that EGF/TGFalpha and the alpha3beta1 integrin, pp60c-src, EGFR and PI3K pathways are mainly associated with the cell spreading of the restitution process during healing of human gastric epithelial wounds.
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Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Transdução de Sinais , Cicatrização/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Mucosa Gástrica/ultraestrutura , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fator de Crescimento Transformador alfa/metabolismoRESUMO
BACKGROUND: Necrotizing enterocolitis (NEC) is the most frequent life-threatening gastrointestinal disease experienced by premature infants in neonatal intensive care units. The challenge for neonatologists is to detect early clinical manifestations of NEC. One strategy would be to identify specific markers that could be used as early diagnostic tools to identify preterm infants most at risk of developing NEC or in the event of a diagnostic dilemma of suspected disease. As a first step in this direction, we sought to determine the specific gene expression profile of NEC. METHODS: Deep sequencing (RNA-Seq) was used to establish the gene expression profiles in ileal samples obtained from preterm infants diagnosed with NEC and non-NEC conditions. Data were analyzed with Ingenuity Pathway Analysis and ToppCluster softwares. RESULTS: Data analysis indicated that the most significant functional pathways over-represented in NEC neonates were associated with immune functions, such as altered T and B cell signaling, B cell development, and the role of pattern recognition receptors for bacteria and viruses. Among the genes that were strongly modulated in neonates with NEC, we observed a significant degree of similarity when compared with those reported in Crohn's disease, a chronic inflammatory bowel disease. CONCLUSIONS: Gene expression profile analysis revealed a predominantly altered immune response in the intestine of NEC neonates. Moreover, comparative analysis between NEC and Crohn's disease gene expression repertoires revealed a surprisingly high degree of similarity between these two conditions suggesting a new avenue for identifying NEC biomarkers.
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Doença de Crohn/complicações , Doença de Crohn/genética , Enterocolite Necrosante/complicações , Enterocolite Necrosante/genética , Perfilação da Expressão Gênica , Transdução de Sinais/genética , Antivirais/metabolismo , Feminino , Humanos , Imunidade Inata/genética , Recém-Nascido , Masculino , Gravidez , Reprodutibilidade dos Testes , Análise de Sequência de RNARESUMO
OBJECTIVES: Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor involved in the regulation of serum glucose and lipid levels. Several HNF4A gene variants have been associated with the risk of developing type 2 diabetes mellitus. However, no study has yet explored its association with insulin resistance and the cardiometabolic risk in children. We aimed to investigate the relationship between HNF4A genetic variants and the presence of metabolic syndrome (MetS) and metabolic parameters in a pediatric population. DESIGN AND METHODS: Our study included 1,749 French-Canadians aged 9, 13 and 16 years and evaluated 24 HNF4A polymorphisms that were previously identified by sequencing. RESULTS: Analyses revealed that, after correction for multiple testing, one SNP (rs736824; P<0.022) and two haplotypes (P1 promoter haplotype rs6130608-rs2425637; P<0.032 and intronic haplotype rs736824-rs745975-rs3212183; P<0.025) were associated with the risk of MetS. Additionally, a significant association was found between rs3212172 and apolipoprotein B levels (coefficient: -0.14 ± 0.05; P<0.022). These polymorphisms are located in HNF4A P1 promoter or in intronic regions. CONCLUSIONS: Our study demonstrates that HNF4α genetic variants are associated with the MetS and metabolic parameters in French Canadian children and adolescents. This study, the first exploring the relation between HNF4A genetic variants and MetS and metabolic variables in a pediatric cohort, suggests that HNF4α could represent an early marker for the risk of developing type 2 diabetes mellitus.
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Fator 4 Nuclear de Hepatócito/genética , Síndrome Metabólica/epidemiologia , Síndrome Metabólica/genética , Polimorfismo de Nucleotídeo Único , População Branca/genética , Adolescente , Glicemia/metabolismo , Canadá/epidemiologia , Canadá/etnologia , Criança , Feminino , Haplótipos , Humanos , Insulina/sangue , Lipídeos/sangue , Masculino , Síndrome Metabólica/sangueRESUMO
The present review focuses on the control of gastrointestinal (GI) tract development. The first section addresses the differences in general mechanisms of GI development in humans versus rodents, highlighting that morphogenesis of specific digestive organs and the differentiation of digestive epithelia occur not only at different stages of ontogeny but also at different rates. The second section provides an overview of studies from the author's laboratory at the Université de Sherbrooke pertaining to the development of the human fetal small intestine and colon. While both segments share similar morphological and functional characteristics, they are nevertheless modulated by distinct regulatory mechanisms. Using the organ culture approach, the author and colleagues were able to establish that hormones and growth factors, such as glucocorticoids, epidermal growth factor, insulin and keratinocyte growth factor, not only exert differential effects within these two segments, they can also trigger opposite responses in comparison with animal models. In the third section, emphasis is placed on the functional development of human fetal stomach and its various epithelial cell types; in particular, the glandular chief cells responsible for the synthesis and secretion of gastric enzymes such as pepsinogen-5 and gastric lipase. Bearing in mind that limitations of available cell models have, until now, greatly impeded the comprehension of molecular mechanisms regulating human gastric epithelial cell functions, the last section focuses on new human gastric epithelial cell models recently developed in the author's laboratory. These models comprise a novel primary culture system of human fetal gastric epithelium including, for the first time, functional chief cells, and human gastric epithelium cell lines cloned from the parental NCI-N87 strain. These new cells lines could serve important applications in the study of pathogenic action and epithelial regeneration.
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Sistema Digestório/crescimento & desenvolvimento , Hormônios Gastrointestinais/fisiologia , Substâncias de Crescimento/fisiologia , Animais , Células Cultivadas , Colo/embriologia , Epitélio/fisiologia , Humanos , Intestinos/embriologia , Roedores/crescimento & desenvolvimento , Estômago/embriologiaRESUMO
OBJECTIVES: The proprotein convertase subtillisin/kexin type 9 (PCSK9) regulates cholesterol metabolism via degradation of low-density lipoprotein receptor (LDLr). Although PCSK9 is abundantly expressed in the intestine, limited data are available on its functions. The present study aims at determining whether PCSK9 plays important roles in cholesterol homeostasis and lipid transport in the gut. METHODS AND RESULTS: Caco-2/15 cells were used allowing the exploration of the PCSK9 secretory route through the apical and basolateral compartments corresponding to intestinal lumen and serosal circulation, respectively. The output of PCSK9 occurred through the basolateral membrane, a site characterized by the location of LDLr. Co-immunoprecipitation studies indicated an association between PCSK9 and LDLr. Addition of purified recombinant wild type and D374Y gain-of function PCSK9 proteins to the basolateral medium was followed by a decrease in LDLr concomitantly with the accumulation of both forms of PCSK9. Furthermore, the latter caused a significant enhancement in cholesterol uptake also evidenced by a raised protein expression of cholesterol transporters NPC1L1 and CD36 without changes in SR-BI, ABCA1, and ABCG5/G8. Moreover, exogenous PCSK9 altered the activity of HMG-CoA reductase and acylcoenzyme A: cholesterol acyltransferase, and was able to enhance chylomicron secretion by positively modulating lipids and apolipoprotein B-48 biogenesis. Importantly, PCSK9 silencing led to opposite findings, which validate our data on the role of PCSK9 in lipid transport and metabolism. Moreover, PCSK9-mediated changes persisted despite LDLr knockdown. CONCLUSIONS: These findings indicate that, in addition to its effect on LDLr, PCSK9 modulates cholesterol transport and metabolism, as well as production of apo B-containing lipoproteins in intestinal cells.
Assuntos
Colesterol/metabolismo , Regulação Enzimológica da Expressão Gênica , Metabolismo dos Lipídeos , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Transporte Biológico , Células CACO-2 , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Células HEK293 , Células Hep G2 , Homeostase , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Intestinos/citologia , Intestinos/enzimologia , Lipoproteínas/metabolismo , Pró-Proteína Convertase 9RESUMO
The intestinal epithelium is a highly dynamic tissue undergoing constant and rapid renewal. It consists of a functional villus compartment responsible for terminal digestion and nutrient absorption and a progenitor cell compartment located in the crypts that produce new cells. The mechanisms regulating cell proliferation in the crypt, their migration, and differentiation are still incompletely understood. Until recently, normal human intestinal cell models allowing the study of these mechanisms have been lacking. In our laboratory, using fetal human intestines obtained at mid-gestation, we have generated the first normal human intestinal epithelial crypt-like (HIEC) cell line and villus-like primary cultures of differentiated enterocytes (PCDE). In this chapter, we provide a detailed description of the methodologies used to generate and characterize these normal intestinal crypt and villus cell models.
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Células Epiteliais/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Cultura Primária de Células/métodos , Proliferação de Células , Separação Celular , Enterócitos/citologia , Enterócitos/metabolismo , Células Epiteliais/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , HumanosRESUMO
Our understanding of gastric epithelial physiology in man is limited by the absence of normal or appropriate cancer cell lines that could serve as an in vitro model. Research mostly relied on primary culture of gastric epithelial cells of animal species, enriched with surface mucous cells, and devoid of glandular zymogenic chief cells. We successfully applied a new nonenzymatic procedure using Matrisperse Cell Recovery Solution to dissociate the entire epithelium from human fetal stomach. Cultures were generated by seeding multicellular aggregates prepared by mechanical fragmentation. We further demonstrate that this simple and convenient technique allows for the maintenance of heterogenous gastric epithelial primary cultures on plastic without a biological matrix as well as the persistence of viable chief cells able to synthesize and secrete gastric digestive enzymes, i.e., pepsinogen and gastric lipase. In wounding experiments, epithelial restitution occurred in serum-reduced conditions and was modulated by exogenous agents. This culture system is thus representative of the foveolus-gland axis and offers new perspectives to establish the influence of individual growth factors and extracellular matrix components as well as their combinatory effects on gastric epithelium homeostasis.
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Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Cultura Primária de Células/métodos , Separação Celular/métodos , Dissecação/métodos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feto , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologiaAssuntos
Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Mucosa Gástrica/citologia , Biomarcadores , Células Cultivadas , Técnicas de Cultura , Matriz Extracelular/química , Feminino , Feto , Mucosa Gástrica/enzimologia , Humanos , Imuno-Histoquímica , Lipase/metabolismo , Pepsina A/metabolismo , GravidezRESUMO
BACKGROUND: Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules. METHODOLOGY/PRINCIPAL FINDINGS: The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 microM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities. CONCLUSIONS/SIGNIFICANCE: Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases.