Dissertations / Theses on the topic 'Modèle intestinal in vitro'
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1
Ponce, de Leon Rodriguez Maria del Carmen. "Développement d’un modèle in vitro d’inflammation intestinale par l’utilisation de lignées cellulaires humaines en co-culture pour l’étude des interactionsavec les micro-constituants alimentaires." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTG009/document.
Abstract:
L’épithélium intestinal, siège de l’absorption des (micro)-nutriments est aussi le premier système de défense de l’organisme. Un déséquilibre dans l’homéostasie peut être à l’origine d’une réaction inflammatoire associée à des défauts de la barrière intestinale et de la fonction immunitaire, ainsi qu’une malabsorption des nutriments, comme rencontré dans les MICI (Maladies Inflammatoires Chroniques de l’Intestin), dans les stratégies de fortification en micronutriments et les pathologies non transmissibles (obésité). Il est donc important de trouver des moyens d’action, via l’alimentation par exemple, pour prévenir ou au minima réduire, les conséquences nutritionnelles et pathologiques de l’inflammation intestinale, et de comprendre les mécanismes impliqués. Parmi les modèles d’études de l’intestin, les modèles in vitro de culture cellulaire sont de plus en plus utilisés et permettent d'évaluer les mécanismes moléculaires d'une manière simple et reproductible et de réduire l'expérimentation animale.Dans ce contexte et dans le but d’étudier l’interaction de composés bioactifs de l’alimentation avec l’intestin en état d’inflammation, le premier objectif de ce travail de thèse a été la mise au point d’un modèle in vitro d’intestin enflammé associant en co-culture deux lignées intestinales humaines : les Caco-2 TC7 (entérocytes) et HT29-MTX (cellules caliciformes) et une lignée immunitaire de macrophages (THP1). Plusieurs marqueurs d’inflammation ont été évalués et nous avons pu montrer que le modèle de tri-culture répondait à un stimulus inflammatoire (LPS/IFNγ), par une augmentation de la production de cytokines pro-inflammatoires (TNF-α, IL6 et IL8) et d’enzymes (INOS et COX2) ainsi que l’expression de leurs gènes. Par ailleurs, une augmentation de la perméabilité épithéliale via une altération des jonctions serrées (TJs) a également pu être mise en évidence ainsi qu’une surproduction de mucus, lesquels sont des caractéristiques reconnus d’inflammation.Le deuxième objectif était d’étudier l’interaction de la β-cryptoxanthine (BCX), caroténoïde des agrumes, lipophile et anti-oxydant, avec le modèle enflammé. Nous avons utilisé pour solubiliser la BCX deux types de micelles (artificielles et physiologiques) et étudié les marqueurs d’inflammation. Bien qu’il semble d’après les résultats préliminaires que les micelles de BCX montrent une tendance à diminuer la production de certaines cytokines (IL6 et IL8), le rôle des constituants des micelles (Tween 40 ou sels biliaires/phospholipides) dans ce phénomène observé et dans la perméabilité épithéliale reste à clarifier par la suiteThe intestinal epithelium, main place of the absorption of (micro)-nutrients is also the first body's defense system. An imbalance in homeostasis can lead to an inflammatory reaction associated with defects in the intestinal barrier and immune function as well as malabsorption of nutrients, as seen in IBD (Inflammatory Bowel Diseases), in micronutrient fortification strategies and noncommunicable diseases (obesity). It is therefore important to find ways of action, for example through diet, to prevent or at least reduce the nutritional and pathological consequences of intestinal inflammation, and to understand the mechanisms involved. Among intestinal models, in vitro cell culture models are increasingly used and allow to evaluate the molecular mechanisms in a simple and reproducible way and to reduce animal experimentation.In this context and in order to study the interaction of dietary bioactive compounds with the intestine in state of inflammation, the first objective of this work was the development of an in vitro model of inflamed intestine combining in co-culture two human intestinal cell lines: Caco-2 TC7 (enterocytes) and HT29-MTX (goblet cells) and an immune cell line of macrophages (THP1). Several inflammation markers were evaluated and we were able to show that the tri-culture model responded to an inflammatory stimulus (LPS / IFNγ), by increasing the production of pro-inflammatory cytokines (TNF-α, IL6 and IL8) and enzymes (INOS and COX2) as well as the expression of their genes. In addition, an increase of epithelial permeability via tight junctions (TJs) alteration has also been demonstrated, as well as overproduction of mucus, which are recognized inflammation characteristics.The second objective was to study the interaction of β-cryptoxanthin (BCX), a lipophilic and antioxidant carotenoid of citrus, with the inflamed model. To solubilize BCX, we used two types of micelles (artificial and physiological) and studied markers of inflammation. Although it appears from the preliminary results that BCX micelles show a tendency to decrease the production of some cytokines (IL6 and IL8), the role of micelle constituents (Tween 40 or bile salts / phospholipids) in the phenomenon observed and in the epithelial permeability remains to be therefore clarified
2
Roy, Isabelle. "Contribution à la mise en place d'un modèle "in vitro" prédictif de l'absorption et du métabolisme intestinal." Paris 5, 1994. http://www.theses.fr/1994PA05P159.
3
Fleury, Mickaël. "Impact de traitements antibiotiques sur la flore digestive du porcelet : Etude in vivo et développement d'une approche en système de fermentation in vitro." Thesis, Rennes 1, 2015. http://www.theses.fr/2015REN1B002/document.
Abstract:
Dans le contexte de l'antibiorésistance, l'objet de ce doctorat vise à évaluer l'impact d'antibiotiques sur le microbiote intestinal de porcelets. La colistine et le ceftiofur, pour lesquels les résistances incluent essentiellement et respectivement mutations chromosomiques et gènes plasmidiques, ont été utilisés. La colistine a significativement réduit la population des entérobactéries, mais aucun E. coli résistant n'a été détecté. L'administration de ceftiofur a eu un impact limité sur les populations bactériennes composant l'écosystème digestif mais a conduit à une forte sélection et à la diffusion d'un gène plasmidique codant pour une bêta-lactamase à spectre étendu. Puis, dans le cadre de la réglementation visant à diminuer l'expérimentation animale, un modèle in vitro colique porcin, nommé PigutIVM, a été mis au point afin de simuler l'environnement digestif du porcelet et a permis de confirmer, in vitro, l'effet observé in vivo de la colistine sur le microbiote. Cet outil a ensuite été utilisé pour évaluer l'impact d'un probiotique, Saccharomyces cerevisiae, comme alternative aux antibiotiques. Le modèle PigutIVM devrait se positionner comme un outil de prédiction pertinent dans les domaines d'investigation aussi bien nutritionnels que pharmacologiquesIn the context of antibiotic resistance, the aim of the current PhD work is to assess the impact of antibiotics on intestinal microbiota of piglets. Two antibiotics i.e. colistin and ceftiofur, for which the main resistances include respectively chromosomal mutations and plasmid genes have been used. Colistin significantly reduced the population of Enterobacteriaceae, but there was no selection of resistant E. coli. The administration of ceftiofur had a limited impact on the bacterial populations that make up the digestive ecosystem but it led to strong selection and dissemination of a plasmid gene encoding an extended-spectrum beta-lactamase. Then, in the framework of regulations to reduce animal testing, an in vitro model of colonic pig named PigutIVM was developed in order to simulate the digestive environment of the piglet and then check the effect of colistin on the microbiota simulated in PigutIVM in vitro. Therefore both the approaches i.e. in vivo and in vitro were compared in order to check the effect of colistin on intestinal microbiota of piglets. This tool was then used to evaluate the impact of a probiotic i.e. Saccharomyces cerevisiae, as alternative to antibiotics. Therefore we assume that this PigutIVM model should be positioned as a relevant predictive tool in the fields of nutritional and pharmacological investigations
4
Altay, Gizem. "Towards the development of biomimetic in vitro models of intestinal epithelium derived from intestinal organoids." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/664864.
Abstract:
Intestinal epithelium is highly specialized tissue organized into crypt-villus units relevant for their effective barrier function and nutrient absorption. In the crypt units reside the proliferative intestinal stem cells (ISCs) that divide and differentiate while migrating along the villi to generate the epithelium. The proliferation, migration and differentiation of ISCs is governed by the tightly controlled spatio-chemical gradients of ISC niche factors; bone morphogenic protein (BMP), wingless/Int (Wnt) and epidermal growth factor (EGF) pathway modulators. In vitro models of the intestinal epithelium, for the most part, based on culturing of intestinal stem cells/crypts in 3D cultures forming structures called organoids. These structures faithfully recapture diverse cell populations and their multicellular organization of native intestinal epithelium. However, 3D closed geometry of intestinal organoids prevents access to the apical region of the epithelium, making them unsuitable for conventional functionality assays. Experimental modeling of intestinal epithelial biology and physiology are limited due to the lack in vitro platforms that recapitulate these key aspects of the small intestinal epithelium: its distinct cell populations, 3D architecture and the gradients of ISC niche biochemical factors along the crypt-villus axis.
Here, we describe development of in vitro models of intestinal epithelium obtained from intestinal organoid-derived crypts. First, we present a method that takes the advantage of substrate stiffness to dictate the formation of monolayers with accessible lumen rather than 3D organoids with a closed geometry. The 2D intestinal epithelium model has in vivo-like crypt-villus cellular organization with all major epithelial cell types and show physiologically relevant tissue barrier function. Then, we describe the development of a more complex model of intestinal epithelium by incorporating a 3D villus-like basement membrane substitute fabricated on hydrogels. For that, poly(ethylene glycol) diacrylate (PEGDA) hydrogels are chosen due to their highly tunable chemical, and mechanical properties, porosity and photocrosslinkable nature allowing easy microstructuring. The formation of 3D bullet-like complex shapes was achieved by photolithography-based crosslinking of PEGDA, a simple, cost-effective approach. The bioactive functionalization of otherwise inert PEGDA for cell adhesion, was achieved by copolymerizing it with acrylic acid and a variety of cell adhesion proteins can be covalently anchored to the 3D villus-like hydrogels. We establish the optimal conditions for the growth of intestinal organoid-derived epithelial monolayers and demonstrated that organoid-derived intestinal epithelial cells successfully formed epithelial monolayers on collagen type I functionalized 3D villus-like PEGDA-acrylic acid hydrogels. Finally, we describe methods to create spatiotemporal gradients of biochemical ISC niche factors on 3D villus-like hydrogels and demonstrate that these gradients can be used to compartmentalize the differentiated epithelial cells. The spatio-chemical gradients of ISC niche biochemical factors on PEGDA hydrogels with proper porosity were successfully generated based on the free diffusion of the factors from a source to a sink chamber in a custom-made microfluidic device allocating the hydrogel and visualized with light-sheet fluorescence microscopy. In silico models were developed to simulate the spatio-chemical gradients formed within the hydrogels. The 3D villus-like PEGDA hydrogels were fabricated on porous membranes and successfully adapted to Transwell® inserts that permitted access to both sides of the hydrogel and the generation of spatio-chemical gradients. The gradients generated in this fashion can be used to compartmentalize the differentiated epithelial cells more towards the tips of the villus-like microstructures.
The 3D villus-like platform improves the current models in providing cells with physiologically representative topographical and mechanical cues and biochemical gradients. Due to its utility, this platform might find uncountable applications. It can be used for the understanding of the basic biology of the intestinal epithelium. In addition, it can be used to culture human intestinal stem cells allowing for the screening of novel therapies and disease modeling.El epitelio intestinal es un tejido altamente especializado, organizado en unidades de criptas y vellosidades que son relevantes para sus eficaces funciones de barrera y absorción de nutrientes. En las unidades de criptas residen las células madre intestinales (ISC) proliferativas que se dividen y diferencian mientras migran a lo largo de las vellosidades, las cuales generan el epitelio maduro. En el epitelio maduro, las ISC y las células proliferativas se localizan en las criptas y las células absorbentes y secretoras diferenciadas en las vellosidades. La proliferación, migración y diferenciación de las ISC se rigen por los gradientes químicos espaciales altamente controlados de los factores de nicho de la ISC; Moduladores de la vía de bone morphogenic protein (BMP), wingless/Int (Wnt) y epidermal growth factor (EGF). El modelado experimental de la biología y la fisiología del epitelio intestinal está limitado debido a la falta de plataformas in vitro que recapitulan estos aspectos clave del epitelio del intestino delgado: sus distintas poblaciones celulares, la arquitectura 3D y los gradientes de factores bioquímicos de nicho ISC a lo largo del eje cripta-vellosidad. Aquí, describimos el desarrollo de modelos in vitro de epitelio intestinal obtenidos de criptas derivadas de organoides intestinales. En primer lugar, presentamos un método para obtener monocapas epiteliales intestinales 2D con lumen accesible y función de barrera fisiológica. A continuación, describimos el desarrollo de andamios biomiméticos 3D similares a vellosidades en hidrogeles de diacrilato de polietilenglicol (PEGDA) utilizando un enfoque fotolitográfico simple y rentable. Demostramos que nuestra plataforma de vellosidades sintéticas apoya la formación de monocapas epiteliales de células epiteliales intestinales derivadas de organoides. Finalmente, describimos métodos para crear gradientes espaciotemporales de factores nicho bioquímicos ISC en hidrogeles 3D similares a vellosidades y demostramos que estos gradientes se pueden usar para compartimentar las células epiteliales diferenciadas. La plataforma 3D que recrea las vellosidades intestinalesmejora los modelos actuales al proporcionar a las células las señales topográficas y mecánicas y los gradientes bioquímicos fisiológicamente representativos. Debido a su utilidad, esta plataforma puede encontrar innumerables aplicaciones. Puede ser utilizada para la comprensión de la biología básica del epitelio intestinal. Además, se puede utilizar para cultivar células madre intestinales humanas que permitan la detección de nuevas terapias y el modelado de enfermedades.
5
Gérémie, Lauriane. "Development of an in-vitro intestinal model featuring peristaltic motion." Thesis, Sorbonne université, 2019. http://accesdistant.sorbonne-universite.fr/login?url=http://theses-intra.upmc.fr/modules/resources/download/theses/2019SORUS118.pdf.
Abstract:
Le Gut-on-chip fait partie d'un thème de recherche plus générale, appelez Organ-on-chip qui a pour objectif de développer des modèles in-vitro qui récapitulent des caractéristiques essentielles de l'organe d'intérêt. Dans le cas de l'intestin, les Gut-on-chip plateformes ont été principalement développés pour reconstituer soit l'architecture 3D de l'intestin, soit sa dynamique et plus particulièrement le péristaltisme. Durant ma thèse j'ai développé une nouvelle et polyvalente Gut-on-chip, présentant ces deux aspects du micro-environnement intestinale. Cette Peristalsis-on-chip nous a permis d'étudier l'influence du mouvement péristaltique sur le comportement cellulaire en fonction de la géométrie de la structure. Pour cette étude nous avons ensemencé des cellules Caco2 sur des substrats 2D ou 3D recouvert de laminine et les avons soumis à un étirement cyclique (à 0.2 Hz et 10\%) pendant 2, 5, 8, 16, 24 et 48 heures. Lors de ces expériences nous avons pu observer une réorientation cellulaire perpendiculaire à l'axe d'étirement que nous avons caractérisé en fonction des conditions de recouvrement, de la confluence initiale, du temps d'étirement et de la géométrie de la structure. Il est intéressant de noter que la réponse cellulaire la plus importante a été obtenue par la combinaison de la géométrie 3D et de l'étirement, ce qui illustre bien le besoin de ces deux éléments pour mieux mimer les conditions intestinales in vivoMy PhD work is part of the organ-on-chip field, and more precisely part of the gut-on-chip field. It is in line with the main objective of this field, which is the development of in-vitro models recapitulating as faithfully as possible the intestinal micro-environment. Through my PhD work I first developed a versatile gut-on-chip platform recapitulating the intestinal 3D architecture as well as its dynamic micro-environment. Therefore, this platform allows us to study the influence of the intestinal dynamic, especially the peristalsis, on cellular behavior in function of the 3D architecture of the scaffold. For this study Caco2 cells have been seeded either on a 2D or a 3D scaffold coated with laminin and submitted to a cyclic stretching (at 0.2 Hz and 10%) for 2, 5, 8, 16, 24 and 48 hours. Our main observation was the cellular reorientation induced by the stretching, therefore we characterized the cell behavior in function of the coating condition, the initial confluency, the stretching time and the scaffold geometry. Interestingly, the strongest cellular response was obtained when the 3D geometry and the stretching was combined illustrating the need of these two stimuli to better mimic the intestinal in vivo conditions
6
Garcia, Rodriguez Alba. "The applicability of in vitro models of the intestinal barrier for the risk assessment of engineered nanomaterials used as food additives." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/669883.
Abstract:
Los avances en el campo de la nanotecnología han permitido desarrollar una gran diversidad de nanomateriales sintetizados artificialmente (NMs), los cuales presentan nuevas y prometedoras aplicaciones en diversas industrias. Debido a sus exclusivas propiedades, los NMs son utilizados en la comida o envoltorios de comida como mejora de textura, color, sabor, estabilizador, etc.
A pesar de sus propiedades innovadoras, existe un aumento en la preocupación sobre si las nanopartículas (NPs) de dióxido de titanio (TiO2) puedan llegar a producir efectos adversos en la salud humana. La Agencia Internacional para la Investigación del Cáncer (IARC) clasificó el TiO2 como posible carcinógeno humano (grupo 2B) debido a suficientes evidencias científicas indicando que las NPs de TiO2 pueden causar cáncer de pulmón a través de su inhalación. Sin embargo, en el mismo informe no se obtuvo resultados concluyentes respecto a la exposición de dichas NPs por vía oral debido a la falta de ensayos toxicológicos e información. Es por eso que el objetivo de la presente Tesis es estudiar de manera in vitro los efectos, factores y mecanismos biológicos que la exposición a NPs metálicas puedan causar en el epitelio del intestino delgado humano.
Para este propósito, se desarrolló por primera vez en nuestro laboratorio un modelo epitelial in vitro que mimetiza el intestino delgado humano. En nuestro primer estudio, se definieron y caracterizaron condiciones de cultivo del ya descrito modelo, Caco- 2/HT29/Raji-B. Según nuestros resultados en los estudios de integridad y permeabilidad, confirmamos que la mejor ratio de células Caco-2 (enterocitos) y HT29 (células calciformes) era 90:10, respectivamente. Paralelamente se detectó la inducción de células presentadoras de antígenos o también conocidas cómo células M, y se propuso un listado de genes cómo marcadores para bio-monitorizar la correcta diferenciación celular y formación de la barrera intestinal in vitro. Finalmente se testó la funcionalidad de nuestro modelo epitelial in vitro exponiéndolo durante 24 h tanto a NPs de TiO2 como de SiO2. Utilizando microscopía laser confocal se demostró que las NPs de TiO2 podrían conllevar efectos adversos en el epitelio intestinal ya que tienen la capacidad de internalizar en las células, llegando incluso, a entrar en contacto con el núcleo celular.
Debida a la gran diversidad de NMs que actualmente se pueden sintetizar artificialmente, y a que cada uno de ellos puede presentar propiedades distintas y por ende afectar de forma diferente sobre la salud humana, el segundo objetivo de la presente Tesis fue valorar los efectos de tres formas distintas de TiO2 (nano-esferas, nano-óvalos i nano-filamentos) utilizando el modelo intestinal Caco-2/HT29. Nuestros resultados demostraron que las tres formas de TiO2 son capaces de desestabilizar el epitelio intestinal, cruzar la cubierta de mucosa, e internalizar en las células hasta alcanzar al núcleo celular. Teniendo en cuenta las imágenes obtenidas con microscopía láser confocal, se demostró que tanto las nano-esferas cómo los nano-óvalos traspasan la barrera intestinal intracelularmente mientras que los nano-filamentos lo hacen por vía paracelular. Finalmente, utilizando el ensayo del cometa, detectamos que las tres NPs produjeron un leve pero estadísticamente significativo daño genotóxico general pero no daño genotóxico oxidativo.
Por último, el tercer estudio se llevó a cabo en el departamento de Ingeniería Biomédica de la Universidad de Binghamton (Binghamton, NY, USA) con el propósito de una mención internacional de la presente Tesis. Puesto que la absorción de nutrientes es una de las principales funciones del intestino delgado, en este estudio se evaluó la actividad de tres enzimas digestivas (Fosfatasa Alcalina Intestinal, Aminopeptidasa-N y la bomba de sodio/potasio) tras exponer el modelo Caco-2/HT29-MTX a NPs de TiO2 y SiO2. Con el fin de simular estrictamente las condiciones reales del tracto gastrointestinal humano, las NPs fueron digeridas de manera artificial simulando el proceso de digestión humano (boca, estómago, intestino), y co-cultivadas con bacterias comúnmente encontradas en el primer segmento del intestino delgado humano, el duodeno. Concretamente se utilizaron el comensal grampositivo Lactobacillus rhamnosus GG, conocido por su actividad probiótica, y el oportunista gramnegativo Escherichia coli NCTC 9001. En este estudio se observó que la presencia de ambas bacterias en el modelo in vitro Caco-2/HT29-MTX, disminuía los efectos adversos de las NPs sobre la actividad enzimática del epitelio.Nano-technological approaches are allowing the development of deliberately engineered nanomaterials (ENMs), presenting promising new applications for many industrial fields. Especially, ENMs possess unique properties and novel uses in food or food packaging materials such as the enhancement of texture, colour, flavour, nutrient stability and food packaging safety. Despite their innovative properties, there is an increasing concern about the possibility that human exposure to TiO2NPs may lead to significant adverse health effects. The International Agency for Research on Cancer (IARC) classified TiO2 as a human carcinogen group 2B because there was enough evidence that nano-TiO2 may cause lung cancer by inhalation. Although oral exposure was also debated by IARC, the final report was inconclusive due to non-existing standardized procedures for nano- TiO2 risk assessment. Due to the potential adverse effects of this ENMs and the lack of information regarding toxicological aspects over the oral exposure, in this Thesis we have carried out in vitro studies on the biological effects of TiO2NPs. For the aforementioned purpose, we set up and characterized, for the first time in our laboratory, an epithelial in vitro model that closely mimics the human small intestine. Thus, in our first study, we defined the best culture conditions for the alreadydescribed model, Caco-2/HT29/Raji-B. From our integrity and permeability findings, we confirmed that the best Caco-2/HT29 cell ratio is 90:10, respectively, as TEER values, paracellular LY permeability and the mucus shed formed correlated well with other studies. We also were able to detect the induction of M-like cells by TEM. Moreover, in order to monitor the proper barrier formation, we proposed a set of genes related to the cell junctional complexes, brush border enzymes, mucus shed components and M-cell markers. Finally, we tested the goodness of our epithelial in vitro model by exposing it to both TiO2NPs and SiO2NPs for 24 h. Our confocal results evidenced the potential adverse effects of TiO2NPs and SiO2NPs on the intestinal epithelium, as NPs internalization and NPs-cell nucleus interaction were observed. Because of the heightened interest in the identification, validation and standardization of the effects associated to exposures to new ENMs, our second study aimed to assess the effects of three different shapes of TiO2NPs (spheres, rods and wires) on the Caco-2/HT29 barrier. Our results demonstrated that the three types of TiO2NPs have the ability to impair the membrane’s integrity, translocate through the mucus shed and internalize in the cells, reaching the nucleus. Taking into account our confocal images results, we hypothesize that due to their shapes, nano-wires are more likely to cross paracellularly, while nano-spheres and nano-rods used intracellular passage to cross the intestinal epithelium. Despite previous evidence that relate the capability of TiO2NPs to produce ROS, we have not detected oxidatively DNA damage. However, and in accordance with the confocal images showing a great amount of NPscell nucleus events, we detected a slight but significant general DNA damage in the barrier’s cells. Finally, the third study was performed under the framework of an international mention carried out in the Biomedical Engineering Department at the Binghamton University (Binghamton, NY, USA). Nutrient absorption is one of the main and most important functions of the small intestine. Thus, to understand and evaluate whether ENPs can trigger physiological potential pathologies, the activity of the intestinal alkaline phosphatase (IAP), aminopeptidase-N (APN) and Na+/K+ ATPase enzymes were measured after exposing the Caco-2/HT29-MTX barrier to TiO2NPs and SiO2NPs for 4 h. Moreover, and in order to further mimic the physiological conditions of a real digestion, the Caco-2/HT29-MTX barrier was exposed to both NPs previously digested and co-cultured with both Escherichia coli and Lactobacillus rhamnosus, as examples of commensal microbiota.
7
SUNDARAM, TAMIL SELVI. "ESTABLISHING IN VITRO INTESTINAL EPITHELIAL CELL MODELS IN TRANSLATIONAL ANIMAL NUTRITION." Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/944348.
Abstract:
Immunomodulatory nutrients as the omega-3 polyunsaturated fatty acids (n-3 PUFA)
and citrus pectins (CPn) are reported to beneficially affect the host intestinal immunity.
However so far, the biological pathways modulated by these nutrients in intestinal
inflammation at the level of intestinal epithelial layer (IEL) remains elusive. To bridge this knowledge gap, the aim of our present study was set in the direction to delineate the effects of n-3 PUFA in porcine IPEC-J2 cell line under (LPS) stress conditions underpinning pig nutrition, combining the state-of-the-art cell-based assays and bioinformatic analysis. The second part of our study was directed towards establishing a primary intestinal epithelial cell (IEC) culture from chicken embryos for the assessment of CPn against LPS stress, underpinning poultry nutrition. Utilizing different cell-based assays, we have successfully demonstrated the proliferative effects of n-3 PUFAs as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the IPEC-J2 cells. Besides, n-3 PUFA pre-treatment (DHA:EPA, 1:2, 10 µM, 24 h) was shown to counteract the cellular damage elicited by different stress factors as LPS, hydrogen-peroxide (H2O2) and dextran sulphate sodium (DSS). In addition, through system biology and multi-omics integration (transcriptomics and proteomics), we have demonstrated the cytoprotective properties of n-3 PUFA in IPEC-J2 cells against LPS-induced inflammatory
and metabolic damage. Specifically, in these cells, we have identified that n-3 PUFA regulate the biological process as, (i) Axon guidance for developmental process; (ii) Defensin and interferon-mediated antimicrobial defense response for homeostasis; (iii) Cell junction assembly under stress-related cell proliferation; (iv) Amelioration of TLR/MyD88 and cytokine signaling in innate immune response; (v) Fatty acid storage in lipid droplets for lipid homeostasis; (vi) Recovery of central carbon metabolic process from dysregulation under infection; (vii) Lipolysis of fatty acids stored in lipid droplets for prevention of cell starvation during infection. To the best of the knowledge, this is the first study to comprehensively map the bioactivity of n-3 PUFA in enterocytes using multi-omics approach. The outcome of the present study, will enable us to better understand the role of n-3 PUFA in intestinal barrier for planning nutritional or
therapeutic strategies. Further in the chicken in vitro study, we have preliminarily demonstrated a simplified method of cell isolation and establishment of primary IEC culture from chicken embryos using mechanical tissue disruptions method. We have also shown that the response of chicken cells is in accordance with the reference IPEC-J2 line using a dose-response study with CPn and LPS. This primary IEC culture model can further be utilized as a starting point for setting up poultry in vitro studies on intestinal barrier.
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Kratz, Jadel Müller. "Implementação e aplicação do modelo in vitro com células Caco-2 para estudo da permeabilidade intestinal de fármacos." Florianópolis, SC, 2011. http://repositorio.ufsc.br/xmlui/handle/123456789/95611.
Abstract:
Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2011Made available in DSpace on 2012-10-26T03:54:33Z (GMT). No. of bitstreams: 1 294811.pdf: 4760452 bytes, checksum: fa236ca094f2d6addc744e0ad2b1cb85 (MD5)
A absorção oral de um fármaco é controlada fundamentalmente por dois fatores: a solubilidade aquosa/dissolução e a permeabilidade intestinal. Portanto, a determinação dessas características ainda nas fases de descoberta e desenvolvimento de fármacos pode prover moléculas com ótimo perfil biofarmacêutico. Nesse sentido, esta tese teve dois objetivos: implementar e validar o modelo de avaliação da permeabilidade in vitro com células Caco-2 no Laboratório de Virologia Aplicada da UFSC, e aplicar esse modelo na determinação da permeabilidade de fármacos e compostos em estudo. Na implementação do modelo Caco-2 foi demonstrada a adequação morfológica das células, através do monitoramento da resistência elétrica transepitelial e da permeabilidade do Lucifer yellow. Através da avaliação da permeabilidade de fármacos marcadores (aciclovir, carbamazepina, hidroclorotiazida, propranolol, vimblastina e verapamil) em experimentos de transporte bi-direcional foi demonstrado que o modelo foi devidamente validado, já que foi estabelecida correlação entre a permeabilidade in vitro e a absorção em humanos. Adicionalmente, uma metodologia por CLAE-UV foi desenvolvida e validada para a determinação concomitante de todos esses fármacos marcadores. Na segunda etapa, avaliou-se o complexo talidomida:hidroxipropil-?-ciclodextrina, desenvolvido e caracterizado no estado sólido por diferentes técnicas, que comprovaram a complexação do fármaco e a redução da sua cristalinidade. Essas alterações culminaram em um leve aumento da solubilidade aparente do fármaco, bem como propiciaram um perfil de dissolução superior, quando comparado ao da talidomida isolada. No entanto, nenhuma alteração na permeabilidade foi detectada. Esses resultados sugerem que esta complexação poderia aumentar a biodisponibilidade oral da talidomida, através do aumento da sua solubilidade nos fluídos gastrointestinais, bem como facilitando a sua dissolução a partir de uma forma farmacêutica sólida. Também foi avaliado o composto anti-herpético galato de pentila, e foi demonstrado que ele apresenta perfis de permeabilidade intestinal e cutânea favoráveis para sua absorção oral e permeação tópica, respectivamente. Assim, após administração oral, sua biodisponibilidade não seria limitada pela permeabilidade intestinal, e no que se refere à administração tópica, ele ficaria restrito às camadas da pele no local de aplicação. Esses dados fornecem informações valiosas para o desenvolvimento de formas farmacêuticas e para a avaliação da atividade antiviral in vivo. Em suma, o modelo com células Caco-2 foi implementado, validado e aplicado satisfatoriamente, o que permitirá a execução de estudos colaborativos, sobretudo com o enfoque do Sistema de Classificação Biofarmacêutica.
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Langan, Laura. "Fish intestinal cultures for ecotoxicological studies : in vitro and primary culture models." Thesis, University of Plymouth, 2017. http://hdl.handle.net/10026.1/9486.
Abstract:
Ecotoxicity testing of chemicals for environmental risk assessment is an area where a high number of vertebrates are used across a variety of industrial sectors. The application of the 3Rs in toxicity testing using fish address both the ethical and societal concerns around this issue in addition to the increasing legislative requests for the incorporation of animal alternatives. This thesis aims to highlight the potential of 3D cell culture models to "bridge the gap" between in vitro and in vivo screening procedures for testing of chemicals with the potential to persist or bioaccumulate thereby improving the predictive power of screening procedures. This thesis examines two alternative methods for their potential use as an intestinal based toxicokinetic tool for environmental risk assessment, utilising an in vitro fish cell line replacement tool (RTgutGC). In addition, for the first time a new intestinal primary cell culture based model was developed to address both intestine region specific response (pyloric, anterior, mid and posterior) and size related adaptability to toxins. Paramagnetic oximetry was used to measure oxygen content within 3D structures (spheroids) in order to better understand the microenvironment of these culture models. Using histology, immunohistochemistry, transepithelial electrical resistance (TEER), transmission electron microscopy (TEM), metabolic, fluorescence and gene expression assays, the comparability of this system to native intestinal response was established. Following exposure to carefully chosen environmental contaminants (Benzo[a]pyrene and Copper), the RTgutGC cell line demonstrated comparable responses to existing literature in terms of uptake, metabolism, DNA damage and the presence an equivalent saturable level. Primary enterocytes cultured on transwell inserts remained viable for upto six weeks, with permeability and metabolic activity comparable to native tissue (both in vitro and ex vivo). Taken in combination, these features of enterocytes represent a profile more closely representative of the intestine then the widely used "gut sac" method. With the potential advantages of incorporating complexity at differing levels (connective tissue layer, intestinal bacteria biome), the intestinal models described offer the potential to screen highly persistent toxins which may require prolonged incubation, in addition to the exploration of complex experimental designs which minimise animal usage (uptake, depuration, uptake). As a consequence, the models developed within this thesis significantly enrich the emerging fish based in vitro testing strategies.
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Deschamps, Charlotte. "Impact du poids corporel et d'une perturbation antibiotique sur le microbiote intestinal du chien : simulation in vitro et stratégies de restauration." Electronic Thesis or Diss., Université Clermont Auvergne (2021-...), 2023. http://www.theses.fr/2023UCFA0055.
Abstract:
Différentes tailles de chiens sont associées à des variations de la physiologie digestive, principalement liées au colon et à ses microorganismes résidents. Ce microbiote intestinal joue un rôle clé en santé, soutenant les processus nutritionnels, immunologiques et physiologiques. Néanmoins, les maladies ou l'antibiothérapie peuvent altérer l'équilibre microbien et induire un état perturbé appelé dysbiose. Pour restaurer l'eubiose du microbiote, de nouvelles stratégies de restauration ont été développées telles que les pré, pro ou postbiotiques. Cependant, peu d'études ont évalué leurs effets sur le microbiote dans le cadre de l'antibiothérapie. Cette thèse entre l'unité Microbiologie, Environnement digestif et Santé de l'Université Clermont Auvergne et les deux sociétés Lallemand Animal Nutrition et Dômes Pharma, visait à étudier l'impact du poids corporel et des antibiotiques sur le microbiote colique canin, ainsi que le potentiel de stratégies de restauration microbienne, à l'aide de modèles intestinaux in vitro.Cette thèse a commencé par évaluer l'impact de différentes méthodes de stockage des échantillons fécaux (congélation 48 h à -80°C, 48 h à -80°C avec du glycérol ou lyophilisation avec maltodextrine/tréhalose) sur la cinétique de colonisation du microbiote et ses activités métaboliques dans Mucosal Artificial Colon (M-ARCOL). Par rapport aux selles fraîches, l'inoculation avec des selles congelées brutes est apparue comme la meilleure option. Grâce à une revue de la littérature, le modèle a été adapté pour reproduire les paramètres nutritionnels, physicochimiques et microbiens spécifiques des conditions du petit, moyen et grand chien dans un nouveau modèle appelé Canine M-ARCOL (CANIM-ARCOL), validé avec des comparaisons in vitro-in vivo. Ceci a permis de reproduire in vitro l'augmentation de la production des principaux acides gras à chaîne courte (AGCC), et la prolifération des Bacteroidota et Firmicutes observées in vivo. Puis, le modèle a permis de réaliser une étude mécanistique, révélant que les paramètres nutritionnels et physicochimiques façonnent l'activité du microbiote associé à la taille du chien, mais que l'inoculum fécal est nécessaire pour reproduire sa composition. Enfin, notre modèle a été adapté pour reproduire la dysbiose induite par les antibiotiques. Conformément aux données in vivo, l'antibiothérapie a induit la prolifération des Enterobacteriaceae, Streptococcaceae et Lactobacillaceae, tandis que la diversité et la production d'AGCC diminuaient. Des effets similaires mais moindres ont été observés dans le microbiote mucosal. Enfin, nous avons évalué l'effet de Saccharomyces boulardii CNCM I-1079 et de Lactobacillus helveticus HA-122 tyndallisée sur la résistance du microbiote pendant le traitement antibiotique et la résilience après celui-ci. Les deux stratégies ont réduit la prolifération des Enterobacteriaceae pendant l'antibiothérapie et permis au cours des deux premiers jours, une résilience plus rapide de la composition et l'activité du microbiote, dans le lumen et le mucus.Ce travail a fourni des informations pionnières et significatives sur l'impact de la taille du chien et de l'antibiothérapie sur la composition et l'activité du microbiote luminal et mucosal du colon canin, comblant les lacunes dans ces domaines. Ces travaux améliorent la compréhension de la résilience du microbiote en réponse aux perturbations antibiotiques. Dans un futur proche, en accord avec les règles européennes 3R visant à réduire les expérimentations animales, nos approches in vitro pourraient être utilisées pour des études mécanistiques des interactions entre nutriments, additifs alimentaires ou produits vétérinaires et microbiote. De telles expériences pourraient être réalisées en situations saines ou perturbées (obésité, maladies inflammatoires de l'intestin ou entéropathies chroniques), en tenant compte des variabilités interindividuelles pour progresser vers une nutrition et une médecine personnaliséesDifferent dog sizes are associated with variations in digestive physiology, mainly related to the large intestine and its resident microorganisms. This gut microbiota plays a key role in animal health, supporting nutritional, immunological and physiological processes. Nevertheless, diseases or antibiotherapy can disturb microbial equilibrium and induce a perturbated state called dysbiosis. To restore microbiota eubiosis, new restorations strategies have been developed such as pre-, pro- or postbiotics. However, very few studies have evaluated their effects on gut microbiota in the context of antibiotherapy. This joint PhD between the Microbiology, Digestive Environment and Health unit from Université Clermont Auvergne and the two compagnies Lallemand Animal Nutrition and Dômes Pharma, aimed to investigate the impact of body weight and antibiotic disturbance on canine colonic microbiota, as well as the potential of microbial restoration strategies, using in vitro gut models.This thesis started by evaluating the impact of different methods for faecal sample storage (48-h freezing -80°C, 48-h -80°C with glycerol or lyophilization with maltodextrin/trehalose) on the kinetics of microbiota colonization and metabolic activities in the Mucosal Artificial Colon (M-ARCOL). Compared to fresh stools, inoculating with raw frozen stool without cryoprotectant was the best option among those tested. Second, thanks to a large literature review, the M-ARCOL model was adapted to reproduce the main nutritional, physicochemical and microbial parameters specific from small, medium and large size conditions in a new model called Canine M-ARCOL (CANIM-ARCOL), further validated through in vitro-in vivo comparisons. This adaptation allowed to reproduce in vitro the increase in Bacteroidota and Firmicutes abundances and higher main short-chain fatty acid (SCFA) concentrations observed in vivo. Then, we used the CANIM-ARCOL to perform a mechanistic study, which revealed that nutritional and physicochemical parameters are enough to shape microbiota activity according to dog size, but faecal inoculum was necessary to reproduce size-related microbiota composition. The next step was to adapt the CANIM-ARCOL to diseased situation, focusing on antibiotic-induced dysbiosis. In accordance with in vivo data, antibiotherapy induced an increase in Enterobacteriaceae, Streptococcaceae and Lactobacillaceae relative abundances while alpha-diversity and SCFA production decreased. Similar but lower effects were observed in mucus-associated microbiota. Lastly, we evaluated the effect of the live probiotic yeast Saccharomyces boulardii CNCM I-1079 and the heat-inactivated bacteria Lactobacillus helveticus HA-122 on microbiota resistance during antibiotic treatment and resilience afterwards. Of interest, both microbial strategies decreased the Enterobacteriaceae bloom during antibiotherapy and allowed, in the first two days, a quicker recovery of microbiota composition and activity, in both the luminal and mucosal compartments.This PhD work provided pioneering and significant insights into the impact of dog size and antibiotherapy on canine colonic luminal and mucus-associated microbiota composition and activity, filling gaps in knowledge in these fields. This work also contributed to a better understanding of microbiota resilience in response to antibiotic disturbance. In a near future, in accordance with the European 3R's rules aiming to reduce at a maximum animal experiments, our in vitro approaches could be used for mechanistic studies on the interactions between nutrients, feed additives or veterinary products and canine colonic microbiota. Such experiments could be performed under healthy but also disturbed gut microbial situations (including obesity, inflammatory bowel diseases or chronic enteropathies), always considering interindividual variabilities to move towards personalized nutrition and medicine
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Dorier, Marie. "Impact du colorant alimentaire E171 et de nanoparticules de dioxyde de titane sur des modèles cellulaires, in vitro, d'épithélium intestinal." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAV082/document.
Abstract:
Les particules de dioxyde de titane (TiO2) sont utilisées dans de nombreux secteurs industriels du fait de leurs propriétés physiques et chimiques intéressantes. Depuis une dizaine d’années, elles sont également utilisées sous forme nanoparticulaire car la taille nanométrique leur apporte de nouvelles propriétés, recherchées dans certaines applications industrielles. Elles sont par exemple utilisées comme colorant blanc dans le secteur de la cosmétologie, de la pharmacologie et dans les industries agroalimentaires. Dans ces dernières, l’utilisation de ces particules est autorisée car le TiO2 est un composé insoluble et relativement inerte. Le colorant alimentaire E171, autorisé depuis 1966, est ainsi constitué de particules de TiO2, initialement sous forme micrométrique, mais il s’avère que selon les procédés de fabrication, entre 10 et 43 % (selon les études) de ces particules présentent un diamètre inférieur à 100 nm, i.e. sont sous forme nanométrique. Ce n’est pas un nanomatériau du point de vue de la définition européenne, il n’est donc pas soumis à l’obligation d’étiquetage dans les produits alimentaires. Le E171 est présent dans de nombreux aliments sans que son impact sur la santé humaine, après ingestion, n’ait été clairement documenté. De plus en plus d’études s’intéressent à la toxicité des nanoparticules (NPs) après leur ingestion, mais peu d’entre elles ont été menées avec le E171 à proprement parler. Les études in vivo et in vitro publiées à ce jour démontrent que les NPs de TiO2 sont peu toxiques. Leur absorption intestinale et leur translocation vers le système sanguin puis des organes secondaires est faible. Les principaux effets décrits sont une augmentation des espèces réactives de l’oxygène associées à un stress oxydant, l’induction de marqueurs de l’inflammation, et plus récemment l’induction du stress du réticulum endoplasmique. Des effets sont également rapportés sur différents paramètres de la barrière intestinale, i.e. le microbiote, le mucus, les transporteurs membranaires, les jonctions cellulaires et l’immunité intestinale. Chez certaines personnes, cette barrière est compromise, elles sont donc potentiellement plus sensibles aux micros et nanos-particules contenues dans l’alimentation. Leur épithélium intestinal est enflammé, et à long terme, ces personnes peuvent développer des maladies inflammatoires chroniques de l’intestin et dans les cas les plus graves, des cancers.L’objectif de cette thèse est d’étudier la toxicité du colorant alimentaire E171 et d’approfondir les connaissances relatives à l’impact des NPs de TiO2 sur le système gastro-intestinal. Pour cela, nous avons travaillé avec différents modèles cellulaires d’épithélia intestinaux humain, un modèle d’épithélium jointif composé d’entérocytes Caco-2, un modèle d’épithélium sécrétant une couche de mucus, composé de cellules Caco-2 et HT29-MTX et enfin un modèle d’épithélium bordant les plaques de Peyer, composé des cellules Caco-2(C1) et RajiB. Ces modèles cellulaires ont été exposés de façon aigüe (6 h, 24 h et 48 h) ou chronique (21 jours), au colorant E171 ainsi qu’à deux NPs de TiO2 : A12, qui a la même structure cristalline que le E171 et P25, une NP très documentée dans la littérature. Nos résultats montrent que le E171 et les NPs de TiO2 sont modérément toxiques, ils n’engendrent pas de mortalité cellulaire ni de dommages à L’ADN. Néanmoins, ils provoquent une accumulation d’espèces réactives de l’oxygène intracellulaires et modulent certains marqueurs impliqués dans le stress oxydant, le stress du réticulum endoplasmique et l’inflammation. Ils impactent également la sécrétion et la composition de la couche de mucus, l’expression des transporteurs ABC, qui sont des paramètres impliqués dans la fonction de barrière de l’épithélium intestinal, le rendant possiblement plus vulnérable aux agressions extérieuresMicro-sized titanium dioxide (TiO2) particles are used for years by industrials for their attractive physical and chemical properties. The use of TiO2 nanoparticles (NPs) is also constantly increasing, because the nanometric size gives new interesting properties to particles which industrials are looking for. In some daily-life products including paints, plastics, paper, medicines and food, micro-sized TiO2 particles are used as a pigment for their opacifying and whitening capacities. The use of TiO2 as a food additive, i.e. E171 in the EU, has been authorized in most countries since the 60ies, without any established acceptable daily intake, because of their low toxicity and intestinal absorption. However, it was recently shown that E171 can contain up to 43% of particles with diameter ranging from 1 to 100 nm, i.e. NPs. Still, E171 is not a nanomaterial as described in the European recommendation of definition because it contains less than 50% of NPs (in number). Food grade TiO2 is present in a wide range of food products while little is known about its toxicological impact to human health. The toxicity of ingested TiO2, either nano- or micro-sized, is increasingly documented, still E171 itself is rarely used in these studies.According to in vivo and in vitro studies, TiO2 particles were proven relatively safe for intestinal cells, no cytotoxicity neither genotoxicity were reported. Nevertheless, particles were often reported to increase reactive oxygen species (ROS) cell content, to impair autophagic processes and modulate gene expression and the content of proteins involved in oxidative stress, endoplasmic reticulum stress and inflammatory response regulation. Interestingly, their reported impact on intestinal cells suggests alteration of almost all the components of the intestinal barrier function, i.e. microbiota, mucus, cell junctions and transporters. This intestinal barrier function is altered in patients suffering from intestinal bowel diseases, these persons are thus possibly more sensitive to mineral particulate in food.The present study aimed at improving knowledge on the toxicity of food-grade TiO2. To this purpose, the impact of E171 was evaluated on in vitro cell models representative of the human intestinal epithelium, i.e. a model of differentiated Caco-2 enterocytes, a model of mucus-secreting epithelium obtained by coculture of Caco-2 and HT29-MTX mucus-secreting cells and a model of the follicle-associated epithelium, which lines Peyer patches, obtained by coculture of Caco-2(C1) and RajiB cells. These cell models were either acutely exposed for 6 h, 24 h and 48 h or chronically exposed for 21 days to E171. In parallel, they were exposed to two model TiO2-NPs, A12 which has the same crystalline structure as E171 and P25, a well-documented TiO2-NPs. Our results show that E171 and TiO2-NPs induced no overt cell mortality but significant oxidative stress, and that they oxidatively damage DNA. They modulate the expression of genes involved in oxidative stress and endoplasmic reticulum stress regulation. They also modulate the expression of genes, as well as the content of proteins from mucus, ABC transporters and inflammatory markers, which are the main players of the intestinal barrier function and presumably increase epithelium sensitivity to xenobiotics. These data suggest that they may be implicated in the development or aggravation of inflammatory bowel diseases
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Cinquin, Cécile Françoise. "Développement et validation d'un nouveau modèle de fermentation colique in vitro avec cellules immobilisées." Thesis, Université Laval, 2005. http://www.theses.ulaval.ca/2005/22769/22769.pdf.
Abstract:
Le microbiote intestinal est un écosystème complexe jouant un rôle très important dans la santé humaine. L’établissement du microbiote intestinal s’effectue dans la première année de vie. Afin de stimuler chez les enfants nourris avec des préparations infantiles les bifidobactéries et lactobacilles, les laits infantiles pourraient être supplémentés avec des probiotiques ou des prébiotiques. Cette supplémentation pourrait leur apporter une protection renforcée contre les troubles intestinaux, les infections et les risques allergiques. Les études in vivo chez les enfants étant difficiles à réaliser à cause de problèmes d’accessibilité et d’éthique, des outils permettant de faire des études in vitro ont été développés. Les systèmes de fermentation continu sont ceux qui se rapprochent le plus des conditions in vivo. Différents modèles ont été proposés, tous utilisent des bactéries fécales libres alors que la microflore bactérienne est présente à l’état planctonique et sessile dans le côlon. Peu de modèles in vitro ont été proposés pour simuler l’écosystème colique chez l’enfant. Le but de cette étude était donc de développer un nouveau modèle de fermentation colique avec cellules immobilisées pour simuler l’écosystème intestinal chez l’enfant. Nous avons montré qu’une microflore fécale pouvait être immobilisée et que sa diversité bactérienne était conservée au cours de la fermentation en continu. La composition et l’activité métabolique de la microflore bactérienne s’équilibraient en fonction des conditions de fermentation à des niveaux voisins de ceux observés in vivo chez l’enfant. Ensuite un modèle de fermentation en continu à trois réacteurs pour simuler le côlon proximal, distal et transverse chez l’enfant a été développé et validé. Ce nouveau modèle a ensuite permis de comparer les effets d’un prébiotique connu (fructooligosaccharide) et d’un exopolysaccharide produit par L. rhamnosus RW-9595M sur l’écosystème intestinal chez l’enfant. Le fructooligosaccharide testé influençait favorablement l’écosystème intestinal alors que l’exopolysaccharide n’était pas métabolisé par le microbiote intestinal chez les enfants. Ce nouveau modèle avec cellules immobilisées possède l’avantage d’obtenir une grande stabilité à la fois de la composition bactérienne et de l’activité métabolique de la microflore, faisant de ce nouveau modèle de fermentation colique un outil de travail pratique, fiable et facile à opérer.The intestinal microbiota is a complex ecosystem playing a key role in human health. The intestinal microbiota establishment occurred during the first year of life. To stimulate bifidobacteria and lactobacilli in formula fed infants, infant formula could be supplemented with probiotics or prebiotics, food ingredient able to improve human health. This supplementation could provide to them a better protection against gastro-intestinal disorders, infections and allergic risks. In vivo studies are difficult to carry out in infants due to accessibility and ethic problems, then in vitro studies become important. Continuous fermentation systems display the closest conditions to those encountered in vivo. Different fermentation systems have been proposed, all are using free-cell cultures whereas in vivo bacteria are present in colon at planktonic and sessile states. To our knowledge only one in vitro model has been proposed to simulate infant colonic fermentation. The aim of this study was to develop a new in vitro system with immobilized cells to simulate infant colonic ecosystem. First a feasibility study was done, we showed that a complex fecal microflora could be immobilized and the inoculum bacterial diversity was conserved in the ecosystem established in the fermentation system. The bacterial composition and metabolic activities of the microbiota reached an equilibrium specific to the fermentation conditions tested. These equilibriums were closed to those detected in vivo in infant colon. Then a three-stage chemostat using immobilized fecal bacteria was developed and validate to simulate simultaneously, proximal, transverse and distal infant colon conditions. This last in vitro colonic fermentation model was used to compared the effect of a well-known prebiotic (fructooligosaccharide) with an exopolysaccharide produced by L. rhamnosus RW-9595M on the infant colonic microbiota. The fructooligosaccharide used beneficially influenced the bacterial ecosystem whereas exopolysaccharide substrate did not seem to be metabolized by infant colonic microbiota. The main advantage of this system with immobilized cells is the great stability of the bacterial composition and metabolic activities of the microflora established in the model. For this, the in vitro colonic fermentation system with immobilized cells is a very efficient tool, easy to operate and reliable.
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Vila, Vecilla Laura. "Desarrollo de un modelo in vitro de barrera intestinal para la evaluación del riesgo de los nanomateriales." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/398242.
Abstract:
El campo de la nanotecnología está expandiéndose de forma exponencial y con ello la preocupación social sobre los posibles efectos de sus productos (nanomateriales; NMs) en los organismos vivos. Dado que las principales vías de exposición a los NMs son el contacto dermal, la inhalación y la ingestión, la barrera intestinal adquiere gran importancia debido a la presencia de NMs en comida y envases de comida, además de su presencia en muchos otros productos de uso diario. En este contexto, el objetivo de esta Tesis es desarrollar un modelo intestinal formado por células Caco-2 para la evaluación del riesgo de NMs. Las células Caco-2 son células de adenocarcinoma de colon que tienen la capacidad de diferenciarse en enterocitos del intestino delgado. Bajo el marco del proyecto NANoREG, los resultados obtenidos han demostrado que se ha desarrollado con éxito un protocolo robusto para obtener una barrera in vitro intestinal estable y reproducible compuesta por estas células Caco-2 diferenciadas. Usando concentraciones subtóxicas de TiO2NPs, ZnONPs, SiO2NPs, CeO2NPs, AgNPs y MWCNT durante 24 horas, se han realizado diferentes tipos de análisis, a saber: a) evaluación de la integridad y permeabilidad de la monocapa, b) internalización celular, c) translocación de los NMs, d) evaluación del daño genotóxico y d) evaluación de la integridad de la monocapa a través de cambios de la expresión del mRNA. Los resultados tras la exposición a concentraciones subtóxicas no mostraron alteración en la integridad ni en la permeabilidad de la monocapa. Además, se observó internalización de las AgNPs tanto en el citoplasma como en el núcleo, mientras que las TiO2NPs y las CeO2NPs se encontraron sedimentadas en la membrana apical de las células. La capacidad de evaluar la translocación de los NMs a través de la barrera celular resultó ser muy limitada. El TEM-EDX, la microscopía confocal y el ICP-MS produjeron resultados de los que se puede concluir una limitada translocación de TiO2NPs, ZnONPS y CeO2NPS. En la evaluación del daño genotóxico mediante el ensayo del cometa, se observó que sólo las AgNPs son capaces de producir daño oxidativo en el DNA y tan sólo en la concentración más alta analizada (50 μg/mL). En la evaluación de la expresión del mRNA de genes de transportadores celulares (SI y SLC15A1) y de adhesión célula-célula (OCCLUDIN y CLAUDIN2), los resultados mostraron mucha variabilidad entre réplicas y no se encontraron cambios significativos en ninguno de ellos tras la exposición a AgNPs.
Al margen del proyecto europeo y con el fin de evaluar el riesgo de la exposición a NMs a largo plazo, se expusieron las células Caco-2 en su estado no diferenciado a concentraciones subtóxicas de AgNPs durante 6 semanas evaluándose su capacidad de transformación. Estas células mostraron necesitar un tiempo de adaptación en el que disminuyen el ritmo de división debido a la exposición hasta que lo recuperan a lo largo del tratamiento. Tras 6 semanas de exposición, las células Caco-2 expresan algunas características de célula transformada: a) aumentan su nivel de proliferación, b) adquieren la capacidad de crecer en soft-agar, y de promover el crecimiento en soft-agar de otras células tumorales (HCT116), c) aumentan la secreción de MMPs, y d) incrementan su capacidad migratoria. Sin embargo, la exposición no mostró inducir cambios en la expresión de los genes involucrados en EMT, adhesión celular, citoesqueleto o supresores de tumores. A pesar de esto, los resultados globales indicarían que las AgNPs no son seguras en términos de carcinogénesis.The field of nanotechnology is increasing every day. For this reason, the social concern about the possible effects of their products (nanomaterials; NMs) in living organisms is also growing. Since the main routes of exposure to NMs are dermal contact, inhalation, and ingestion, the intestinal barrier is very important due to the presence of NMs in food and food packaging, in addition to its presence in many other daily products. In this context, the objective of this Thesis is to develop an intestinal model formed by Caco-2 cells in order to assess the risk of NMs. Caco-2 cells are from colon adenocarcinoma and have the capacity to differentiate into enterocytes of the small intestine. Under the framework of the NANoREG’s project, the results have shown that we have successfully developed a robust protocol to obtain stable and reproducible in vitro intestinal barrier composed of these differentiated Caco-2 cells. Using sub-toxic concentrations of TiO2NPs, ZnONPs, SiO2NPs, CeO2NPs, AgNPS and MWCNT for 24 hours, we have performed different types of analysis: a) assessment of the integrity and permeability of the monolayer, b) cellular internalization, c) translocation of NMs, d) evaluation of genotoxic damage, d) assessment of the integrity of the monolayer through changes in mRNA expression. The results after exposure to sub-toxic concentrations showed no alteration in the integrity or the permeability of the monolayer. Furthermore, internalization of AgNPS was observed in both the cytoplasm and nucleus while TiO2NPs and CeO2NPs found sedimented on the apical membrane of cells. The ability to evaluate the translocation of NMs through the cell barrier proved to be very limited. TEM-EDX, confocal microscopy, and ICP-MS showed limited translocation of TiO2NPs, ZnONPs, and CeO2NPS. In the evaluation of genotoxic damage by comet assay, it was observed that only AgNPS are capable of producing oxidative DNA damage and only at the highest concentration tested (50 μg/mL). In assessing mRNA expression of genes of cellular transporters (SI and SLC15A1) and cell-cell adhesion (OCCLUDIN and CLAUDIN2), results showed a lot of variability between replicates and no significant changes were found in any of them after exposure to AgNPS. Besides the European project and in order to assess the risk of exposure to NMs in the long term, Caco-2 cells in their undifferentiated state were exposed for 6 weeks to sub-toxic concentrations of AgNPS evaluating its transformation capacity. These cells were subjected to a period of adaptation in which the cells decreased the rate of division due to exposure until regaining it at the end of the treatment. After 6 weeks of exposure, the Caco-2 cells expressed some features of the transformed cells: a) increased the proliferation rate, b) acquired the ability to grow in soft agar, and promoted the growth of other tumour cells (HCT116) in soft-agar, c) increased the secretion of MMPs, and d) increased their migratory capacity. However, exposure didn’t show changes in the expression of genes involved in EMT, cell adhesion, cytoskeleton or tumour suppressors. Despite this, the overall results indicate that the AgNPS are not safe in terms of carcinogenesis.
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Costa, Tie Bezerra. "Efeito da privação da glutamina sobre as células secretoras do epitélio intestinal em um modelo in vitro de enteroide." reponame:Repositório Institucional da UFC, 2012. http://www.repositorio.ufc.br/handle/riufc/10626.
Abstract:
COSTA, Tie Bezerra. Efeito da privação da glutamina sobre as células secretoras do epitélio intestinal em um modelo in vitro de enteroide. 2014. 73 f. Dissertação (Mestrado em Ciências Médicas) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2014.Submitted by denise santos (denise.santos@ufc.br) on 2015-02-13T11:59:15Z No. of bitstreams: 1 2014_dis_tbcosta.pdf: 1905458 bytes, checksum: 3bfbf27d06a97ecac348168074140446 (MD5)
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The intestinal epithelium is formed and sustained by a population of stem cells capable of generating different cell lines while maintaining pluripotent and self-renewal capacity. Glutamine is a conditional essential amino acid important for the maintenance of the intestinal epithelium. However, few studies to date have explored the role of glutamine in the fine regulation of the intestinal crypt cell turnover. In order to evaluate the role of glutamine in the crypt cell turnover, an in vitro enteroid model was used, where stem cells are capable of generating an epithelium containing the main intestinal secretory cell lines (Paneth, goblet, and enteroendocrine cells) and absorptive enterocytes as well, while forming a villus-crypt like structure. This model was used to test the effect of 24h of glutamine deprivation (standard media with 2mM glutamine vs glutamine-free media) on epithelial turnover by counting EdU- labeled cells/total number per section and cell apoptosis by counting cleavage-caspase 3- labeled cells/total number per section. In order to assess crypt secretory function, Paneth and goblet crypt cell ratios (target secretory cell/total cell number per crypt) were measured. The number of Paneth and goblet cells was measured with the aid of confocal microscopy and lysozyme and mucin-2 immunostainning. In addition, Paneth and globet cell product transcripts (lysozyme and mucin, respectively) were measured using quantitative Real-time PCR. In order to assess the potential immunomodulatory role of glutamine, innate immune element transcripts, Toll like receptor and their accessory protein MD-2, and cytokines, as follows: TNF-α, IL-1β and CXCL-1, were measured. Glutamine deprivation reduced the number of EdU positive cell ratio as compared with the enteroid under the standard media (p=0.006). No significant differences regarding Paneth and goblet cell ratios were seen between groups following glutamine deprivation. Glutamine deprivation significantly decreased lysozyme transcripts as compared with the enteroid under the standard media (p=0.007), but not for mucin-2 transcripts, related to goblet cell function. Decreased TNF-α and MD-2 transcription (p=0.005 and p=0.016, respectively) were found following glutamine deprivation. Altogether, our findings reinforce the glutamine positive role on the intestinal epithelial turnover and furthermore suggest an important glutamine regulatory effect over Paneth cells and the innate immune system. The enteroid model provides an important tool the dissect the mechanisms of glutamine protection and shed light for future studies.
O epitélio intestinal é formado e mantido por uma população de células-tronco capaz de gerar diferentes linhagens celulares, mantendo a pluripotência e a capacidade de auto-renovação. A glutamina é um aminoácido essencial condicional importante para a manutenção do epitélio do intestino. No entanto, poucos estudos têm explorado o papel da glutamina na regulação fina do turnover celular da cripta intestinal. Com o propósito de avaliar o papel da glutamina n o turnover de células da cripta, foi utilizado um modelo in vitro de enteroide, onde as células-tronco são capazes de gerar um epitélio contendo as principais linhagens de células secretoras intestinais (célula de Paneth, célula caliciforme e célula enteroendócrina), além dos enterócitos absortivos, com a formação de uma estrutura tipo vilosidade-cripta. Este modelo foi usado para testar o efeito de 24 horas de privação de glutamina (meio padrão com glutamina a 2 mM vs meio livre de glutamina) no turnover epitelial através da contagem de células marcadas por Edu/número total por secção e ainda na apoptose celular, através da contagem de células marcadas para caspase 3-clivada/número total por secção. A fim de avaliar a função secretora das criptas, as razões das células de Paneth e caliciformes por cripta (célula alvo/número total de células secretoras por cripta) foram medidas. O número de células de Paneth e caliciformes foi obtido com o auxílio da microscopia confocal e imunomarcação para lisozima e mucina-2. Além disso, os transcritos dos produtos das células de Paneth e caliciformes (lisozima e mucina, respectivamente) foram analisados utilizando o método de PCR quantitativo em tempo real. Com intuito de avaliar o potencial papel imunomodulador da glutamina, transcritos de elementos da imunidade inata, receptor de tipo Toll e sua proteína acessória MD-2, e citocinas, a saber: TNF-α, IL-1β e CXCL-1, foram medidos. A privação de glutamina reduziu o número de células Edu positivas, em comparação com o enteroide sob meio padrão (p=0,006). Não houve diferença significativa em relação às razões das células de Paneth e células caliciformes entre os grupos após privação de glutamina. A privação da glutamina diminuiu significativamente os transcritos de lisozima, em comparação com o enteroide sob meio padrão (p = 0,007), mas não para mucina-2, transcrição relacionada com a função das células caliciformes. Uma transcrição reduzida para TNF- α e MD-2 (p=0,005 e p=0,016, respectivamente) foi observada após a privação de glutamina. Ao todo, nossos achados reforçam o papel positivo da glutamina sobre o turnover do epitélio intestinal e, além disso, sugerem um importante efeito regulador da glutamina sobre as células de Paneth e resposta imune inata. O modelo enteroide fornece uma ferramenta importante para dissecar os mecanismos de proteção pela glutamina e guiar estudos futuros.
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Alves, Luis Antonio de Oliveira. "ExpressÃo e regulaÃÃo da quinase celular intestinal (ICK) em modelo de desnutriÃÃo in vivo e in vitro." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=12743.
Abstract:
A desnutriÃÃo pode afetar a arquitetura intestinal, causando atrofia da mucosa e comprometendo o turnover epitelial. Embora o intestino seja capaz de responder compensatoriamente à desnutriÃÃo, os mecanismos moleculares pelos quais o intestino responde à privaÃÃo proteica nÃo estÃo completamente esclarecidos. A quinase celular intestinal (ICK) à uma proteÃna altamente conservada do tipo serina/treonina e um novo componente das vias de sinalizaÃÃo que regulam a proliferaÃÃo celular na cripta intestinal. Para avaliar o papel da resposta intestinal compensatÃria à privaÃÃo proteica, regulada pela ICK, foi medida a ativaÃÃo de vias moleculares relacionadas à proliferaÃÃo e sobrevivÃncia celulares, como a via canÃnica Wnt/β-catenina, a via da proteÃna alvo da rapamicina em mamÃferos (mTOR), vias da proteÃna-quinase ativada por mitÃgeno (MAPK) e da proteÃna quinase B (PKB/Akt), bem como a expressÃo de marcadores para cÃlulas-tronco intestinais (LgR-5 e Bmi1) por imunoblotting num modelo in vivo em camundongos fÃmeas C57BL/6J submetidas à uma dieta hipoproteica (contendo 2% de proteÃna) por cinco dias e controles nutridos recebendo dieta padrÃo. TambÃm foi realizado um estudo in vitro utilizando cÃlulas HCT-8 de adenocarcinoma ileocecal humano desafiadas com meio padrÃo com restriÃÃo de soro fetal bovino (0-1%) para avaliar a resposta da ICK na presenÃa ou nÃo de fatores trÃficos intestinais como glutamina (2mM), alanil-glutamina (10 e 50 mM) e zinco (10 e 50μM), alÃm de caseÃna e albumina sÃrica bovina (BSA) na concentraÃÃo de 0,25 e 0,5% no meio, respectivamente. A anÃlise de RNAm foi feita para avaliar o transcrito de ICK por q-PCR, apÃs privaÃÃo proteica. No intuito de avaliar o efeito dessa quinase sobre a proliferaÃÃo celular e apoptose, foi realizado o silenciamento do gene da ICK com uso de RNA de interferÃncia em cÃlulas HCT-8. A viabilidade celular foi avaliada com base na exclusÃo do azul de tripan. Posteriormente, foram realizados testes de western blot para caspase 3 e 9, PARP-clivada, alÃm de anexina V por citometria de fluxo para avaliar apoptose, assim como western blot para via Wnt/β-catenina e ciclina D1 no intuito de medir os mecanismos relacionados à proliferaÃÃo celular. Dessa forma, foi identificado um aumento significativo e transitÃrio nos nÃveis intestinais de ICK na desnutriÃÃo induzida pela raÃÃo hipoproteica, concomitante com a ativaÃÃo de vias moleculares relacionadas à proliferaÃÃo e sobrevivÃncia, bem como o aumento da expressÃo de marcadores para cÃlulas-tronco intestinais. Esse trabalho tambÃm documentou que a
privaÃÃo proteica, induzida por baixa concentraÃÃo de soro, aumenta a expressÃo de ICK em cÃlulas HCT-8, efeito que foi revertido pela adiÃÃo de BSA no meio. O mesmo resultado, nÃo foi observado com outros compostos como glutamina, alanil-glutamina e zinco. Apesar do aumento da expressÃo da ICK apÃs privaÃÃo proteica, nÃo houve diferenÃa significativa da sua transcriÃÃo quando comparado com os controles. O silenciamento do gene de ICK reduziu significativamente a sinalizaÃÃo da via Wnt-β-catenina e ciclina D1 em cÃlulas HCT-8 com aumento da apoptose por um mecanismo dependente de caspases. Os resultados sugerem que o aumento da expressÃo de ICK em resposta à privaÃÃo proteica à um mecanismo de proteÃÃo que limita a apoptose e suporta a proliferaÃÃo celular epitelial na desnutriÃÃo.Malnutrition can affect the intestinal architecture, causing mucosal atrophy and compromising epithelial turnover. Although the gut is able to compensatorily respond to malnutrition, the molecular mechanisms by which the gut responds to protein deprivation are not completely understood. The intestinal cell kinase (ICK) is a highly conserved serine/threonine protein and a novel component of the signaling pathways that regulate cell proliferation in the intestinal crypt. In order to evaluate the role of the intestinal compensatory response to protein deprivation mediated by ICK, the activation of molecular pathways related to cell proliferation and survival were measured in C57BL/6J mice subjected to low protein diet (containing 2% protein) for five days and received standard chow-fed controls. We analyzed the intestinal canonical Wnt/β-catenin pathway, mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK) and protein kinase B (PKB/Akt) by immunoblotting. We also measured the expression of intestinal stem cells markers (LGR-5 and Bmi1). We also conducted an in vitro study using human ileocecal adenocarcinoma cells (HCT-8) starved with low serum (0-1%) to evaluate the ICK response with or without gut trophic factors, such as glutamine (2 mM), alanyl-glutamine (10 and 50mM), and zinc (10 and 50μM), casein and bovine serum albumin (BSA) at a concentration of 0.25 or 0.5% in the medium, respectively. We also assessed ICK mRNA transcript by q-PCR after protein deprivation. In order to evaluate the effect of this kinase on cell proliferation and apoptosis, the ICK gene was silenced using interference RNA in HCT-8 cells. Cell viability was measured by trypan blue exclusion. Furthermore, caspase 3 and 9, cleaved PARP, analyzed by western blot, and annexin V, measured by flow cytometry, were used to assess apoptosis. In order to measure ICK effect on cell proliferation, we analyzed Wnt/β-catenin and cyclin D1 pathways by western blot. We identified a significant and transient increase in ICK intestinal levels following low-protein induced malnutrition, concomitant with the activation of molecular pathways related to proliferation and survival, as well as increased expression of intestinal stem cell markers. This work also documented that the protein deprivation, induced by low serum concentration, increased the expression of ICK in HCT-8 cells, an effect that was reversed by BSA in the medium. This reverse effect was not seen with other compounds such as glutamine, alanyl-glutamine or zinc. Despite the increase in ICK expression after protein deprivation, no significant difference in its transcripts was found, when compared with controls. The silencing of the ICK gene significantly decreased Wnt/β-catenin and cyclin D1 signaling activation in HCT-8 cells with increased apoptosis by a caspase dependent mechanism. Our results suggest that the increased ICK expression in response to protein deprivation is a protective mechanism that limits cell apoptosis and supports epithelial cell proliferation following malnutrition. Keywords: Malnutrition. Protein deprivation. Intestinal cell kinase. β
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Alves, Luis Antonio de Oliveira. "Expressão e regulação da quinase celular intestinal (ICK) em modelo de desnutrição in vivo e in vitro." reponame:Repositório Institucional da UFC, 2014. http://www.repositorio.ufc.br/handle/riufc/15323.
Abstract:
ALVES, Luis Antonio de Oliveira. Expressão e regulação da quinase celular intestinal (ICK) em modelo de desnutrição in vivo e in vitro. 2014. 144 f. Tese (Doutorado em Ciências Médicas) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2014.Submitted by denise santos (denise.santos@ufc.br) on 2016-03-04T13:13:41Z No. of bitstreams: 1 2014_dis_laoalves.pdf: 14635596 bytes, checksum: e4f1330eb5aa6bd3be3b7e58d448a025 (MD5)
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Malnutrition can affect the intestinal architecture, causing mucosal atrophy and compromising epithelial turnover. Although the gut is able to compensatorily respond to malnutrition, the molecular mechanisms by which the gut responds to protein deprivation are not completely understood. The intestinal cell kinase (ICK) is a highly conserved serine/threonine protein and a novel component of the signaling pathways that regulate cell proliferation in the intestinal crypt. In order to evaluate the role of the intestinal compensatory response to protein deprivation mediated by ICK, the activation of molecular pathways related to cell proliferation and survival were measured in C57BL/6J mice subjected to low protein diet (containing 2% protein) for five days and received standard chow-fed controls. We analyzed the intestinal canonical Wnt/β-catenin pathway, mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK) and protein kinase B (PKB/Akt) by immunoblotting. We also measured the expression of intestinal stem cells markers (LGR-5 and Bmi1). We also conducted an in vitro study using human ileocecal adenocarcinoma cells (HCT-8) starved with low serum (0-1%) to evaluate the ICK response with or without gut trophic factors, such as glutamine (2 mM), alanyl-glutamine (10 and 50mM), and zinc (10 and 50μM), casein and bovine serum albumin (BSA) at a concentration of 0.25 or 0.5% in the medium, respectively. We also assessed ICK mRNA transcript by q-PCR after protein deprivation. In order to evaluate the effect of this kinase on cell proliferation and apoptosis, the ICK gene was silenced using interference RNA in HCT-8 cells. Cell viability was measured by trypan blue exclusion. Furthermore, caspase 3 and 9, cleaved PARP, analyzed by western blot, and annexin V, measured by flow cytometry, were used to assess apoptosis. In order to measure ICK effect on cell proliferation, we analyzed Wnt/β-catenin and cyclin D1 pathways by western blot. We identified a significant and transient increase in ICK intestinal levels following low-protein induced malnutrition, concomitant with the activation of molecular pathways related to proliferation and survival, as well as increased expression of intestinal stem cell markers. This work also documented that the protein deprivation, induced by low serum concentration, increased the expression of ICK in HCT-8 cells, an effect that was reversed by BSA in the medium. This reverse effect was not seen with other compounds such as glutamine, alanyl-glutamine or zinc. Despite the increase in ICK expression after protein deprivation, no significant difference in its transcripts was found, when compared with controls. The silencing of the ICK gene significantly decreased Wnt/β-catenin and cyclin D1 signaling activation in HCT-8 cells with increased apoptosis by a caspase dependent mechanism. Our results suggest that the increased ICK expression in response to protein deprivation is a protective mechanism that limits cell apoptosis and supports epithelial cell proliferation following malnutrition. Keywords: Malnutrition. Protein deprivation. Intestinal cell kinase. β
A desnutrição pode afetar a arquitetura intestinal, causando atrofia da mucosa e comprometendo o turnover epitelial. Embora o intestino seja capaz de responder compensatoriamente à desnutrição, os mecanismos moleculares pelos quais o intestino responde à privação proteica não estão completamente esclarecidos. A quinase celular intestinal (ICK) é uma proteína altamente conservada do tipo serina/treonina e um novo componente das vias de sinalização que regulam a proliferação celular na cripta intestinal. Para avaliar o papel da resposta intestinal compensatória à privação proteica, regulada pela ICK, foi medida a ativação de vias moleculares relacionadas à proliferação e sobrevivência celulares, como a via canônica Wnt/β-catenina, a via da proteína alvo da rapamicina em mamíferos (mTOR), vias da proteína-quinase ativada por mitógeno (MAPK) e da proteína quinase B (PKB/Akt), bem como a expressão de marcadores para células-tronco intestinais (LgR-5 e Bmi1) por imunoblotting num modelo in vivo em camundongos fêmeas C57BL/6J submetidas à uma dieta hipoproteica (contendo 2% de proteína) por cinco dias e controles nutridos recebendo dieta padrão. Também foi realizado um estudo in vitro utilizando células HCT-8 de adenocarcinoma ileocecal humano desafiadas com meio padrão com restrição de soro fetal bovino (0-1%) para avaliar a resposta da ICK na presença ou não de fatores tróficos intestinais como glutamina (2mM), alanil-glutamina (10 e 50 mM) e zinco (10 e 50μM), além de caseína e albumina sérica bovina (BSA) na concentração de 0,25 e 0,5% no meio, respectivamente. A análise de RNAm foi feita para avaliar o transcrito de ICK por q-PCR, após privação proteica. No intuito de avaliar o efeito dessa quinase sobre a proliferação celular e apoptose, foi realizado o silenciamento do gene da ICK com uso de RNA de interferência em células HCT-8. A viabilidade celular foi avaliada com base na exclusão do azul de tripan. Posteriormente, foram realizados testes de western blot para caspase 3 e 9, PARP-clivada, além de anexina V por citometria de fluxo para avaliar apoptose, assim como western blot para via Wnt/β-catenina e ciclina D1 no intuito de medir os mecanismos relacionados à proliferação celular. Dessa forma, foi identificado um aumento significativo e transitório nos níveis intestinais de ICK na desnutrição induzida pela ração hipoproteica, concomitante com a ativação de vias moleculares relacionadas à proliferação e sobrevivência, bem como o aumento da expressão de marcadores para células-tronco intestinais. Esse trabalho também documentou que a privação proteica, induzida por baixa concentração de soro, aumenta a expressão de ICK em células HCT-8, efeito que foi revertido pela adição de BSA no meio. O mesmo resultado, não foi observado com outros compostos como glutamina, alanil-glutamina e zinco. Apesar do aumento da expressão da ICK após privação proteica, não houve diferença significativa da sua transcrição quando comparado com os controles. O silenciamento do gene de ICK reduziu significativamente a sinalização da via Wnt-β-catenina e ciclina D1 em células HCT-8 com aumento da apoptose por um mecanismo dependente de caspases. Os resultados sugerem que o aumento da expressão de ICK em resposta à privação proteica é um mecanismo de proteção que limita a apoptose e suporta a proliferação celular epitelial na desnutrição.
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Cornu, Raphaël. "Nanoparticules et santé : de grandes promesses thérapeutiques, mais pour quel risque ?" Thesis, Bourgogne Franche-Comté, 2019. http://www.theses.fr/2019UBFCE018.
Abstract:
Les nanoparticules sont définies comme des structures sphériques dont le diamètre maximum est de 100 nanomètres. Les domaines d’applications des nanoparticules incluant l’industrie agroalimentaire et pharmaceutique, sont extrêmement nombreux. L’Homme peut être quotidiennement exposé à des nanoparticules via différentes voies d’administration (orale, intraveineuse, pulmonaire et cutanée). En raison de leur taille nanométrique, les nanoparticules possèdent des propriétés physico-chimiques uniques, induisant de fortes interactions avec l’environnement biologique. Ces caractéristiques ont été largement exploitées pour la conception de nanomédicaments pour le diagnostic et la thérapie. Cependant, les problèmes liés à leur toxicité ont été mis en lumière parallèlement à leur développement. Ce travail de thèse vise à étudier la toxicité potentielle des nanoparticules. L'évaluation toxicologique a été réalisée à l'aide de modèles cellulaires adaptés aux voies systémique et orale. Les mécanismes impliqués dans la nanotoxicité ont été étudiés afin d’identifier des facteurs de toxicité. La première partie de ce travail a été consacrée à l’étude de la toxicité hépatique in vitro et in vivo des nanoparticules de PLGA et de silice. Les nanoparticules de PLGA sont utilisées comme vecteurs de médicaments alors que les nanoparticules de silice jouent le rôle d’agent antiagglomérant dans l’industrie alimentaire et également d’excipients pharmaceutiques. Leurs effets sur la fonction hépatique et en particulier sur l'activité des cytochromes P450 ont été évalués. La seconde partie de ce travail a consisté à étudier l’impact des nanoparticules de silice sur la barrière intestinale, en particulier sur la perméabilité paracellulaire et l’intégrité de la barrière. En mettant en exergue les différences interespèces ou le rôle protecteur du mucus, ce projet a démontré que le choix des outils toxicologiques était crucial pour une évaluation prédictive de la nanotoxicité. La taille, les propriétés de surface et la composition ont été identifiées comme des facteurs importants de toxicitéNanoparticles are defined as spherical structures with a maximum diameter of 100 nanometers. The application fields of nanoparticles including food and pharmaceutical industries are extremely broad. Human can be exposed daily to nanoparticles through various administration routes (oral, intravenous, pulmonary and cutaneous). Due to their size at the nanoscale, nanoparticles have unique physicochemical, inducing strong interactions with the biological environment. These features were widely exploited for the conception of nanomedicines for the diagnosis and the therapy. However, issues relative to their biological toxicity were addressed in the same time. This thesis work aims to investigate the potential toxicity of nanoparticles. Toxicological evaluation was performed using cell models adapted for the systemic and the oral routes. Mechanisms involved in the nanotoxicity were studied to identify toxicity factors. The first part of the work focused on the in vitro and in vivo hepatic toxicity of PLGA and silica nanoparticles. PLGA nanoparticles are used as drug carriers while silica nanoparticles play the role of anticaking agent in food industry and of pharmaceutical excipients. Their effects on the liver function and especially on the cytochrome P450 activity were investigated. The second part of the work consisted to study the impact of silica nanoparticles on the intestinal barrier, especially on the paracellular permeability and the integrity of the barrier. By emphasizing interspecies differences or the protective role of mucus, this project demonstrated that the choice of toxicological tools was crucial for a predictive nanotoxicity evaluation. Size, surface properties and composition were identified as major toxicity factors
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Roos, Karin Hester. "Effects of plant extracts and phytoconstituents on the intestinal transport of indinavir / K.H. Roos." Thesis, North-West University, 2012. http://hdl.handle.net/10394/9692.
Abstract:
There is a global rise in the use of herbal products in combination with allopathic medicines, while most patients do not inform their health care providers of the use of these natural products. Both pharmacodynamic and pharmacokinetic interactions between herbal products and conventional drugs must be avoided for the wellbeing of the patient. Increasing evidence from in vitro and in vivo studies indicate that changed drug pharmacokinetics by co-administered herbs may be attributed to modulation of efflux drug transporters such as P-glycoprotein (P-gp). Garlic (Allium sativum), lemon (Citrus limonum) and beetroot (Beta vulgaris) are widely used by human immunodeficiency virus (HIV) patients, especially following the pronouncement by a former President of South Africa and the Ministers of Health at that time who promoted the use of these botanicals in HIV patients.
The aim of this study was to measure the bi-directional in vitro transport of indinavir, a protease inhibitor, in the presence of crude extracts and pure phytoconstituents of A. sativum (L-alliin and diallyl disulphide), C. limonum (hesperidin and eriocitrin) and B. vulgaris (betaine monohydrate and ß-carotene) across excised porcine intestinal tissue in Sweetana-Grass diffusion chambers. In the negative control group, the transport of indinavir alone (200 M) was determined with no modulator added. In the positive control group, the transport of indinavir was determined in the presence of verapamil (100 M), a known P-gp related efflux inhibitor. The control experiments were used to indicate that the effects of the test compounds were caused by their action and not by chance interferences or external factors. Samples collected at pre-determined time intervals were analysed by means of a validated high performance liquid chromatography (HPLC) method and the transport was expressed as the apparent permeability coefficient (Papp) and the transepithelial flux (J) from which the efflux ratio (ER) and the net flux (Jnet) values were calculated. Statistical analysis was used to compare the results of the test compounds with the control groups in order to indicate significant differences.
The mean ER value for indinavir in the negative control group was 1.41 ± 0.170 and in the positive control group it was 0.56 ± 0.0426. Statistically significant (p < 0.05) inhibition of indinavir efflux as indicated by reduced ER values was obtained for L-alliin (ER = 0.280 ± 0.030), diallyl disulphide (ER = 0.505 ± 0.034) and ß-carotene (ER = 0.664 ± 0.075). Inhibition of indinavir efflux will lead to increased transport and therefore a potentially higher bioavailability. Statistically significant (p < 0.05) promotion of indinavir efflux as indicated by increased ER values was obtained for C. limonum crude extract (ER = 5.551 ± 0.575) and hesperidin (ER = 3.385 ± 0.477), which potentially may lead to lower bioavalability. B. vulgaris crude extract (p = 0.8452), betaine monohydrate (p = 0.9982), A. sativum crude extract (p = 0.7161) and eriocitrin (p = 0.4431) displayed no statistically significant effect compared to the negative control group on indinavir transport across excised porcine intestinal tissue.
The results from this study demonstrate that L-alliin, diallyl disulphide and ß-carotene have an inhibitory effect on indinavir efflux, which may significantly increase indinavir plasma levels after oral administration. C. limonum crude extract and hesperidin promote indinavir efflux, which may significantly reduce indinavir plasma levels. These pharmacokinetic interactions between certain drugs and plant extracts may negatively affect the anti-retroviral treatment of HIV patients, but deliberate and controlled inclusion of L-alliin, diallyl disulphide and ß-carotene in dosage forms may possibly cause more effective delivery of protease inhibitors after oral administration resulting in less frequent dosing intervals.Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.
19
Cleusix, Valentine. "Study of the effects of Lactobacillus reuteri and reuterin on the human intestinal microbiota using in vitro models /." Zürich : ETH, 2006. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=16976.
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Schweinlin, Matthias Oliver [Verfasser], Heike [Gutachter] Walles, Stefan [Gutachter] Störk, and Beate [Gutachter] Niesler. "Development of advanced human intestinal in vitro models / Matthias Oliver Schweinlin ; Gutachter: Heike Walles, Stefan Störk, Beate Niesler." Würzburg : Universität Würzburg, 2017. http://d-nb.info/1123505942/34.
21
Gérard-Champod, Marie. "Caractérisation et modélisation in vitro de l'écosystème jéjuno-iléal du veau de boucherie." Phd thesis, Université Blaise Pascal - Clermont-Ferrand II, 2009. http://tel.archives-ouvertes.fr/tel-00726333.
Abstract:
Dans le secteur du veau de boucherie, l'interdiction par l'Union Européenne en Janvier 2006 des antibiotiques utilisés comme facteurs de croissance (AFCs) a déclenché la recherche de nouveaux additifs alimentaires permettant de retrouver les performances de croissance des animux et de réduire l'incidence des pathologies gastro-intestinales. Afin de réaliser un screening à grande échelle de ces additifs, l'objectif de ces travaux était de mettre au point un système de fermentation continu modélisant in vitro l'environnement intestinal du veau de boucherie. Les principaux groupes bactériens cultivables et les paramètres majeurs de la composition biochimique du contenu jéjuno-iléal ont tout d'abord été caractérisés in vivo. Ensuite, un système de fermentation in vitro a été mis au point et son milieu nutritif optimisé. Trois milieux nutritifs testés ont conduit à une stabilisation des grand groupes bactériens dénombrés. Le milieu M3 a été choisi pour la suite des expériences en raison de sa composition très proche de celle du contenu jéjuno-iléal des veaux et parce qu'il a conduit à une faible variabilité inter-réplicats. Le modèle a ensuite été utilisé afin d'étudier l'impact, sur la microflore du veau de boucherie, d'un additif alimentaire : le carvacrol. Les résultats de ce premier essai se sont avérés difficilement exploitables car le pouvoir antioxydant du carvacrol en a faussé l'interprétation. Avec l'ajout d'un antioxydant plus fort que le carvacrol dans le milieu nutritif, l'impact du carvacrol sur la microflore devrait pouvoir être analysé. Même si des améliorations restent à effectuer, les premiers résultats sont très prometteurs. Après screening in vitro et choix de nouveaux additifs susceptibles de remplacer les AFCs, des essais in vivo devront être effectués pour valider l'intérêt des substances sélectionnées in vitro. En effet, la modélisation in vitro ne simule pas tous les phénomènes ayant lieu in vivo et reste un outil de "pré-screening" en amont.
22
Costa, Tie Bezerra. "Efeito da privaÃÃo da glutamina sobre as cÃlulas secretoras do epitÃlio intestinal em um modelo in vitro de enteroide." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=13454.
Abstract:
CoordenaÃÃo de AperfeiÃoamento de NÃvel SuperiorO epitÃlio intestinal à formado e mantido por uma populaÃÃo de cÃlulas-tronco capaz de gerar diferentes linhagens celulares, mantendo a pluripotÃncia e a capacidade de auto-renovaÃÃo. A glutamina à um aminoÃcido essencial condicional importante para a manutenÃÃo do epitÃlio do intestino. No entanto, poucos estudos tÃm explorado o papel da glutamina na regulaÃÃo fina do turnover celular da cripta intestinal. Com o propÃsito de avaliar o papel da glutamina n o turnover de cÃlulas da cripta, foi utilizado um modelo in vitro de enteroide, onde as cÃlulas-tronco sÃo capazes de gerar um epitÃlio contendo as principais linhagens de cÃlulas secretoras intestinais (cÃlula de Paneth, cÃlula caliciforme e cÃlula enteroendÃcrina), alÃm dos enterÃcitos absortivos, com a formaÃÃo de uma estrutura tipo vilosidade-cripta. Este modelo foi usado para testar o efeito de 24 horas de privaÃÃo de glutamina (meio padrÃo com glutamina a 2 mM vs meio livre de glutamina) no turnover epitelial atravÃs da contagem de cÃlulas marcadas por Edu/nÃmero total por secÃÃo e ainda na apoptose celular, atravÃs da contagem de cÃlulas marcadas para caspase 3-clivada/nÃmero total por secÃÃo. A fim de avaliar a funÃÃo secretora das criptas, as razÃes das cÃlulas de Paneth e caliciformes por cripta (cÃlula alvo/nÃmero total de cÃlulas secretoras por cripta) foram medidas. O nÃmero de cÃlulas de Paneth e caliciformes foi obtido com o auxÃlio da microscopia confocal e imunomarcaÃÃo para lisozima e mucina-2. AlÃm disso, os transcritos dos produtos das cÃlulas de Paneth e caliciformes (lisozima e mucina, respectivamente) foram analisados utilizando o mÃtodo de PCR quantitativo em tempo real. Com intuito de avaliar o potencial papel imunomodulador da glutamina, transcritos de elementos da imunidade inata, receptor de tipo Toll e sua proteÃna acessÃria MD-2, e citocinas, a saber: TNF-α, IL-1β e CXCL-1, foram medidos. A privaÃÃo de glutamina reduziu o nÃmero de cÃlulas Edu positivas, em comparaÃÃo com o enteroide sob meio padrÃo (p=0,006). NÃo houve diferenÃa significativa em relaÃÃo Ãs razÃes das cÃlulas de Paneth e cÃlulas caliciformes entre os grupos apÃs privaÃÃo de glutamina. A privaÃÃo da glutamina diminuiu significativamente os transcritos de lisozima, em comparaÃÃo com o enteroide sob meio padrÃo (p = 0,007), mas nÃo para mucina-2, transcriÃÃo relacionada com a funÃÃo das cÃlulas caliciformes. Uma transcriÃÃo reduzida para TNF- α e MD-2 (p=0,005 e p=0,016, respectivamente) foi observada apÃs a privaÃÃo de glutamina. Ao todo, nossos achados reforÃam o papel positivo da glutamina sobre o turnover do epitÃlio intestinal e, alÃm disso, sugerem um importante efeito regulador da glutamina sobre as cÃlulas de Paneth e resposta imune inata. O modelo enteroide fornece uma ferramenta importante para dissecar os mecanismos de proteÃÃo pela glutamina e guiar estudos futuros.
The intestinal epithelium is formed and sustained by a population of stem cells capable of generating different cell lines while maintaining pluripotent and self-renewal capacity. Glutamine is a conditional essential amino acid important for the maintenance of the intestinal epithelium. However, few studies to date have explored the role of glutamine in the fine regulation of the intestinal crypt cell turnover. In order to evaluate the role of glutamine in the crypt cell turnover, an in vitro enteroid model was used, where stem cells are capable of generating an epithelium containing the main intestinal secretory cell lines (Paneth, goblet, and enteroendocrine cells) and absorptive enterocytes as well, while forming a villus-crypt like structure. This model was used to test the effect of 24h of glutamine deprivation (standard media with 2mM glutamine vs glutamine-free media) on epithelial turnover by counting EdU- labeled cells/total number per section and cell apoptosis by counting cleavage-caspase 3- labeled cells/total number per section. In order to assess crypt secretory function, Paneth and goblet crypt cell ratios (target secretory cell/total cell number per crypt) were measured. The number of Paneth and goblet cells was measured with the aid of confocal microscopy and lysozyme and mucin-2 immunostainning. In addition, Paneth and globet cell product transcripts (lysozyme and mucin, respectively) were measured using quantitative Real-time PCR. In order to assess the potential immunomodulatory role of glutamine, innate immune element transcripts, Toll like receptor and their accessory protein MD-2, and cytokines, as follows: TNF-α, IL-1β and CXCL-1, were measured. Glutamine deprivation reduced the number of EdU positive cell ratio as compared with the enteroid under the standard media (p=0.006). No significant differences regarding Paneth and goblet cell ratios were seen between groups following glutamine deprivation. Glutamine deprivation significantly decreased lysozyme transcripts as compared with the enteroid under the standard media (p=0.007), but not for mucin-2 transcripts, related to goblet cell function. Decreased TNF-α and MD-2 transcription (p=0.005 and p=0.016, respectively) were found following glutamine deprivation. Altogether, our findings reinforce the glutamine positive role on the intestinal epithelial turnover and furthermore suggest an important glutamine regulatory effect over Paneth cells and the innate immune system. The enteroid model provides an important tool the dissect the mechanisms of glutamine protection and shed light for future studies.
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Batista, Lobo Samira. "Development of 'In vitro' intestinal models to study the pharmacology of drugs affecting the gastrointestinal tract in normal and diseased conditions. Development of a cell culture model for intestinal pharmacology." Thesis, University of Bradford, 2009. http://hdl.handle.net/10454/4295.
Abstract:
Studies investigating the effect of 5-HT receptors mediating a response in the neonatal intestine have been limited. There are evidences that the development of new neurones continues past postnatal term and this suggests that receptors expression may differ during maturation. Thus, 'in vitro' experiments were carried out to investigate the effects of ACh, atropine, 5-HT and its related drugs on intact intestinal segments taken from the ileum of adult and neonate rats. The application of ACh (3nM-1mM) and 5-HT (3nM-1mM) induced contractions in a concentration dependent manner in all tissues examined. The 5-HT induced contractions were only sensitive to antagonism by atropine (1μM) in segments taken from the neonates but not adults. The pre-treatment with methysergide (5-HT1/2/5-7 receptor antagonist), ritanserin (5-HT2 receptor antagonist), granisetron (5-HT3 receptor antagonist) and RS 23597 (5-HT4 receptor antagonist) at 1μM or a combination of ritanserin, granisetron, plus RS 23597 at 1μM significantly reduced or abolished contractile responses induced by 5-HT. SB 269970A (5-HT7 receptor antagonist) and WAY 100635 (5-HT1A receptor antagonist) at 1μM failed to influence contractile responses induced by 5-HT or the challenges to 5-HT receptor agonists, 5-CT (5-HT1A/7 receptor agonist) and 8-OH-DPAT (5-HT1A receptor agonist) at a concentration range of 10nM-0.1mM, indicating the unlikely involvement of 5-HT1A and 5-HT7 receptors in the mediation of contractile responses in the neonatal rat ileum. Results indicate differences in cholinergic receptor involvement during postnatal maturation and suggest the involvement of 5-HT2, 5-HT3 and 5-HT4 receptors in the mediation of contractile responses to 5-HT in the neonatal rat ileum. There is a growing need to decrease animal usage in pharmacological experiments. This may be achieved by the development of 'in vitro' cell culture models. Thus attempts were also made to develop a cell culture model of neonatal intestine to further investigate the action of pharmacologically active agents. The isolation of individual cell populations from segments taken from the intestine of rat neonates were achieved by ligation of both ends of the intestine prior to incubation in trypsin so that a gradual dissociation could be monitored. This was supported by histological procedures, determining the time required to extract large numbers of cells from different intestinal layers. Differential adhesion and selective cytotoxicity techniques were used for further purification of intestinal smooth muscle cells (ISMC), neuronal cells, and a coculture of ISMC and neuronal cells, and these were characterised through immunostaining with antibodies to α-smooth muscle actin, α-actinin and the 5-HT3 receptor. A protocol for cryopreservation of ISMC was designed in order to protect cells against genetic instability, enhance cell availability and reduce animal usage. Results showed that cells extracted from the intestine are viable for up to 4-months. ISMC functionality was analysed via the application of known pharmacologically active drugs on ISMC, which were plated onto glass and silicone elastomer substrate. The cultured ISMC responded to the application of drugs such as potassium chloride (KCl), carbachol, 5-HT and noradrenaline (NA). Large population of cocultures seeded onto silicone elastomers or cholesteric liquid crystal substrates (LC) were assessed for their ability to produce a collective response to KCl application. Attempts were made to detect any deformations of the substrate surface due to the exposure to KCl and NA. Cholesteric LC substrates seemed to be the most suitable material for investigating the cellular tensions. The availability of cell cultures allowed the development of an intestinal model of inflammation. This was achieved through the use of lipopolysaccharide (LPS)-induced inflammation and was confirmed by assessing the levels pro-inflammatory mediators interleukin (IL-8) and nitric oxide (NO), which were significantly elevated. Reduction of IL-8 ad NO was also examined using granisetron and L-NAME and Chaga mushroom extract. Granisetron and L-NAME reduced the NO production during short incubation times. However, an elevated level of NO was observed when longer treatment times were examined. The Chaga mushroom extract caused a significant reduction in NO production in the model of inflammation. This indicates that this model may be a valuable tool for the investigation of other pro-inflammatory mediators and may contribute for the investigation of more selective drugs in the management of intestinal inflammation in neonates.
24
Le, Bacquer Olivier. "Effets trophiques de la glutamine : études in vivo chez le nouveau-né prématuré et in vitro sur un modèle d'entérocytes humains en culture." Nantes, 2002. http://www.theses.fr/2002NANT18VS.
Abstract:
Les études réalisées chez l'animal et chez l'homme suggèrent que, dans les situations de catabolisme protéique, l'apport de glutamine stimule la synthèse protéique au niveau du corps entier et a un effet trophique sur l'intestin. Les mécanismes d'action de la glutamine restent à déterminer. Les résultats montrent que 1) chez le prématuré, une supplémentation en glutamine, réduit la synthèse protéique au niveau du corps entier, mais a un effet anti-catabolique en diminuant à la fois la protéolyse et l'oxydation protéique ; 2) ils montrent également que la carence en glutamine réduit la synthèse protéique. . . . . La supplémentation en glutamine restaure la plupart de ces paramètres, probablement via son métabolisme énergétique. En conclusion, ces données suggèrent que la glutamine pourrait favoriser le gain protéique chez le prématuré par son effet anti-catabolique ; elle pourrait également intervenir dans la trophicité du tube digestif de ces enfants en stimulant la synthèse protéique intestinaleAnimal and human studies suggest that glutamine supply may enhance whole body protein synthesis stimulation and improve gut trophicity during critical illness. The mechanisms of these effects remain unclear. This work combines an in vivo study in very-low-birth-weight infants with two in vitro studies using the Caco-2 cell line as a model of human enterocytes in culture. Our results show that 1) in parenterally fed preterm, a 24h glutamine supplementation decreases whole body protein synthesis, but may have an acute protein-sparing effect, as it suppresses protein breakdown and oxidation; 2) we also demonstrate that glutamine deprivation impairs protein synthesis in a model of enterocytes. Finally, 3) apical nutrient deprivation, a model of fasting, impairs protein synthesis, depletes glutathione pool, and increases transepithelial permeability. Most of these parameters are restored by glutamine supplementation, probably through glutamine utilization as a source of energy. Taken together, these findings suggest that glutamine may improve protein accretion in preterm infants through an anti-catabolic effect, and regulate gut barrier function by stimulating intestinal protein synthesis
25
Vila, Giraut Anna. "Hydrogel co-networks of gelatin methacryloyl and poly(ethylene glycol)diacrylate sustain 3D functional in vitro models of intestinal mucosa." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/670921.
Abstract:
Conventional in vitro cell culture models do not possess the complexity that the native tissues offer. Because of this, the functional properties of the tissues are not properly mimicked, which causes poorly predictive capabilities. Engineered tissues, which combine biofabrication and tissue engineering techniques, try to overcome this gap by providing the cells with an environment similar to the native tissue, recapitulating (I) the physicochemical and mechanical properties of the cellular matrix, (II) the multicellular complexity of the different tissue compartments, and (III) the 3D structures of the tissues. These new engineered models are key factors to improve the platforms for basic research studies, testing new drugs or modelling diseases. Among all the engineered tissues, the intestinal mucosa is not well represented. The intestinal mucosa is formed by the epithelium, which is a multicellular monolayer laying on top of the lamina propria, a connective tissue containing several cell types (mesenchymal cells, immune cells). The gold standard intestinal models are based on epithelial cell lines derived from colon cancer cells grown on the hard porous membranes of the Transwell® inserts. The lack of the intestinal stromal compartment and the growth on a hard surface give high transepithelial electrical resistance and low apparent permeability. Therefore, the development of better in vitro platforms, which integrates both compartments and provides epithelium-lamina propria cell interactions, is highly desirable.
In this work, we describe an easy and cost-effective method to engineer a 3D intestinal mucosa model that combines both the epithelium and the lamina propria compartments. To build the 3D scaffolds we chose hydrogels as materials to mimic the physicochemical and mechanical properties of intestinal tissue. Thus, hydrogel co- networks of gelatin methacryolyl (GelMA), a natural polymer, and poly(ethylene glycol) diacrylate (PEGDA), a synthetic polymer, are photopolymerized. On one hand, GelMA provides biodegradation and cell adhesion sequences but it lacks long-term mechanical stability. On the other hand, PEGDA, is non-biodegradable and does not present cell adhesion motifs. Nevertheless, it has good mechanical properties. By this technique, the lamina propria compartment of the intestinal mucosa can be reproduced in vitro. To do that, GelMA and PEGDA polymers are laden with mesenchymal cells (fibroblasts
or myofibroblasts) and/or immune cells (macrophages). We demonstrated that GelMA
– PEGDA hydrogel co-networks support the growth of these cells and epithelial monolayers on top of the scaffolds. Embedding fibroblasts or myofibroblasts on the hydrogel co-networks enhance the formation and the maturity of the Caco-2 epithelial monolayers, providing barrier properties similar to in vivo. The presence of the stromal cells, also enhances the recovery of the epithelial integrity when the epithelium is temporally damaged. Finally, an immunocompetent model is obtained by the encapsulation of macrophages in the constructs. The presence of macrophages does not influence the formation of the epithelium. However, when the epithelial monolayer is disrupted, the presence of mesenchymal and immune cells in the stromal compartment increases cytokine secretion in a synergistic manner. Our model can successfully mimic the interactions between stromal and epithelial compartments found in vivo intestinal tissue, offering a potential platform to be used to study absorption and toxicity of drugs, as well as cell behaviour under physiological and pathological conditions.En l’intestí prim trobem la mucosa, la capa més externa de la paret intestinal, formada per dos compartiments, la lamina propia i l’epiteli, en els quals resideixen diferents tipus cel·lulars. La interacció entre els dos compartiments és essencial pel funcionament correcte del intestí. Actualment, els models intestinals in vitro es basen en el cultiu de línies cel·lulars epitelials sobre membranes dures i de plàstic, els quals no representen correctament ni la complexitat ni la organització cel·lular trobada en el intestí prim, ja que manquen de la lamina propria. Conseqüentment, els resultats obtinguts utilitzant aquests models són significativament poc fisiològics i no comparables amb els trobats en condicions in vivo (alts valors de resistència elèctrica transepitelial, subestimació dels valors d’absorció de molècules a través de la ruta paracel·lular i alteració en la expressió enzims digestius). Per reduir les distàncies entre els models intestinals in vitro i l’intestí, s’han de desenvolupar plataformes que modelitzin: (I) les propietats fisicoquímiques i mecàniques de la matriu extracel·lular, (II) els dos compartiments de la mucosa intestinal, i si pogués ser (III) l’arquitectura tridimensional. Per aconseguir aquests requeriments, en aquesta tesi s’utilitzen hidrogels formats per polímers naturals (gelatina metacrilada (GelMA)) co-polimeritzats amb polímers sintètics (poly(ethylene glycol) diacrylate (PEGDA)) com a substrat per imitar els dos compartiments intestinals. Per reproduir la lamina propria, els polímers de GelMA i PEGDA es dissolen juntament amb el fotoiniciador. Tot seguit, les cèl·lules residents de la lamina propria (fibroblasts, miofibroblasts o macròfags) es barregen amb la solució de pre-polimer, s’aboca a les piscines de PDMS, s’exposa a llum ultraviolada, i s’obté l’hidrogel amb les cèl·lules en el seu interior. Finalment, les cèl·lules epitelials es sembren a la superfície del hidrogel. En aquesta tesi, s’ha obtingut un hidrogel amb propietats fisicoquímiques i mecàniques similars a la matriu extracel·lular del intestí humà, i que permeten el cultiu cel·lular fins a 21 dies, imitant els dos compartiments. A més, la el co-cultiu de cèl·lules residents de la lamina propria juntament amb les cèl·lules epitelials, ens ha permès obtenir un model 3D de la mucosa intestinal in vitro amb propietats fisiològiques més semblants a la del intestí prim.
26
Maares, Maria Henrietta [Verfasser], Hajo [Akademischer Betreuer] Haase, Hajo [Gutachter] Haase, and Anna [Gutachter] Kipp. "Investigations on zinc resorption using in vitro intestinal models / Maria Henrietta Maares ; Gutachter: Hajo Haase, Anna Kipp ; Betreuer: Hajo Haase." Berlin : Technische Universität Berlin, 2019. http://d-nb.info/1186130229/34.
27
Leonard, Fransisca [Verfasser], and Claus-Michael [Akademischer Betreuer] Lehr. "Novel cell based in vitro models to study nanoparticle interaction with the inflamed intestinal mucosa / Fransisca Leonard. Betreuer: Claus-Michael Lehr." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2013. http://d-nb.info/1052781454/34.
28
THOREUX, KARINE. "Vieillissement de l'appareil digestif et nutrition. Modeles in vitro et in vivo de vieillissement intestinal, impact des laits fermentes sur la trophicite digestive." Paris 7, 1997. http://www.theses.fr/1997PA077303.
Abstract:
Longtemps ignore, le vieillissement de l'appareil digestif doit etre considere dans les intrications entre denutrition, pathologies liees a l'age et le vieillissement lui-meme. Des modeles experimentaux sont necessaires. De plus, les produits laitiers fermentes, ayant de nombreuses proprietes benefiques sur la sante, representeraient des facteurs nutritionnels importants. La lignee iec-6 presente une evolution reproductible au cours des passages de la culture, avec une repartition morphotypique caracteristique, associee a des alterations des parametres de proliferation et des voies de signalisation intracellulaire. Cette lignee representerait un bon modele in vitro des mecanismes de senescence intestinale. Les laits fermentes ont une action trophique sur la lignee iec-6. Ceci est confirme in vivo chez des souris alimentees par un regime supplemente en yaourt ou lait fermente par lactobacillus casei, dont l'intestin grele presente une augmentation de l'aire villositaire, de la proliferation cellulaire, et de l'activite des hydrolases intestinales, et une hyperplasie des clones secreteurs. Les relations entre pancreas exocrine et intestin grele ont ete etudiees au cours du jeune et de la realimentation de rats sprague-dawley ages de 22 mois. Le vieillissement du pancreas exocrine, plus important que celui de l'intestin grele, se traduit par un affaissement majeur de la capacite secretoire d'amylase, associe a une augmentation du taux de cholecystokinine circulante. La denutrition aggrave ce phenomene et les animaux ages montrent une desadaptation a la realimentation. L'intestin grele proximal presente une aire villositaire diminuee, mais une proliferation accrue, tandis que l'intestin distal a des niveaux fonctionnels plus eleves, suggerant une compensation de la digestion par l'ileon. Le rat sprague-dawley de 22 mois est un bon modele de vieillissement in vivo et des interrelations entre pancreas exocrine et intestin grele au cours de la denutrition de la personne agee.
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Neuhoff, Sibylle. "Refined in vitro Models for Prediction of Intestinal Drug Transport : Role of pH and Extracellular Additives in the Caco-2 Cell Model." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis: Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5814.
30
Balandras, Frédérique. "Étude in vitro de la sensibilité de l'[alpha]-casozépine, décapeptide à activité benzodiazépine mimétique, à diverses protéases et peptidases du tractus gastro-intestinal. Étude comportementale chez le rat Wistar de l'activité anxiolytique des fragments F97 et F95 libérés par la pepsine." Thesis, Vandoeuvre-les-Nancy, INPL, 2008. http://www.theses.fr/2008INPL047N/document.
Abstract:
L’[alpha]-casozépine, décapeptide issu de l’hydrolysat trypsique de la caséine [alpha]s1, possède in vitro une affinité pour le site benzodiazépine des récepteurs GABAA centraux (Lecouvey et al., 1997 ; Miclo et al., 2001). Des résultats attestent du potentiel anxiolytique de ce décapeptide seul ou au sein de son hydrolysat, in vivo, en administration intrapéritonéale et per os chez le rat Wistar (Guesdon et al., 2006 ; Miclo et al., 2001 ; Violle et al., 2007) et chez l’Homme en administration orale (Kim et al., 2007 ; Messaoudi et al., 2005). Pour déterminer la sensibilité de l’[alpha]-casozépine à différentes attaques protéolytiques, des cinétiques d’hydrolyses gastriques, pancréatiques et intestinales, ont été réalisées in vitro avec les enzymes et systèmes enzymatiques suivants ; pepsine, trypsine, [alpha]-chymotrypsine, CorolasePP®, vésicules membranaires de bordure en brosse d’entérocytes de rats et épithélium entérocytaire reconstitué par les cellules de la lignée Caco-2. Pour chacune des conditions d’hydrolyses étudiées, l’ensemble des fragments peptidiques libérés ont été séparés par chromatographie liquide haute performance en phase inversée et caractérisés par spectrométrie de masse. Ces analyses mettent en évidence la résistance partielle de l’[alpha]-casozépine à certaines enzymes et systèmes enzymatiques. L’hydrolyse pepsique de l’[alpha]-casozépine libère notamment deux fragments : 91YLGYLEQ97 et 91YLGYL95, nommés F97 et F95 qui s’avèrent être plus résistants que [alpha]-casozépine à certaines enzymes employées. Ainsi pour permettre une meilleure compréhension de la relation entre la structure de l’[alpha]-casozépine et la fonction benzodiazépine mimétique obtenu in vivo, ces peptides tronqués dans leur partie carboxy-terminale ont été testés in vivo chez le rat Wistar en injection intrapéritonéale. Ils s’avèrent être également détenteurs d’activité anxiolytique. Cependant aucun transfert de l’[alpha]-casozépine et des fragments F97 et F95 n’a pu être observé au travers de l’épithélium entérocytaire reconstitué par le modèle cellulaire de la lignée Caco-2 et ce malgré les différentes conditions de cultures et d’analyses testées[alpha]-Casozepine, a tryptic decapeptide from bovine [alpha]s1-casein, have in vitro an affinity for the benzodiazepine site of the central receptor GABAA (Lecouvey et al., 1997; Miclo et al., 2001). Some results display the anxiolytic potential of this peptide alone or within its hydrolysat, in vivo, in intra peritoneal administration and per os in Wistar rats (Guesdon et al., 2006; Miclo et al., 2001; Violle et al., 2007) and in human in oral administration (Kim et al., 2007; Messaoudi et al., 2005). To determine the sensitivity of [alpha]-casozepine in various proteolytic attacks, kinetics of gastric, pancreatic and intestinal hydrolyses, were realized in vitro with enzymes and following enzymatic systems; pepsin, trypsin, [alpha]-chymotrypsin, CorolasePPTm, brush border membranar vesicles rats enterocytes and enterocytes epithelium reconstituted by the Caco-2 cell line. For each of the conditions of studied hydrolysis, all the peptides fragments released were separated by liquid chromatography high-performance in inverted phase and characterized by mass spectrometry. These analyses underline the partial resistance of [alpha]-casozepine in some enzymes and enzymatic systems. [alpha]-Casozepine pepsic hydrolysis release in particular two peptide fragments: 91YLGYLEQ97 and 91YLGYL95, named F97 and F95 who turn out to be more resistant than [alpha]-casozepine in some used enzymes. So to allow a better understanding of the relation between structure of [alpha]-casozepine and the benzodiazepine mimetic function obtained in vivo, these peptides truncated in their carboxy-terminal party were tested in vivo with Wistar rat in intra peritoneal injection. They turn out to be also holders of anxiolytic activity. However no transfer of [alpha]-casozepine and fragments F97 and F95 was observed through the enterocyte epithelium reconstituted by the cellular model Caco-2 and this in spite of the various conditions of cultures and tested analyses
31
Belliard, Anne-Marie. "Influence de l'IL2, de l'INFγ et du TNFα sur l'activité, l'expression et la localisation de la P-glycoprotéine intestinale dans un modèle in vitro : les cellules Caco-2 en culture." Paris 11, 2003. http://www.theses.fr/2003PA114819.
Abstract:
La Pgp est une protéine localisée entre autre dans la bordure en brosse des entérocytes où elle rejette dans la lumière intestinale les xénobiotiques substrats. La susceptibilité à développer une inflammation de l'intestin est liée en partie à une diminution de l'expression de la Pgp. L'IL2, le TNFa et l'IFNg participant au développement de l'inflammation intestinale, notre objectif a été de déterminer les effets spécifiques de chaque cytokine sur l'activité, l'expression et la localisation de la Pgp dans un modèle in vitro : les cellules Caco-2 en culture. L'activité de la Pgp a été mesurée par le transport unidirectionnel de la rhodamine 123, l'expression par RT-PCR semi-quantitative et par Western blot et la localisation par microscopie confocale. Nous avons montré une diminution de l'activité de la Pgp après exposition des cellules à l'IL2 et au TNFa et l'absence d'effet de l'IFNg sur l'activité bien qu'il entraîne une modification de la répartition de la Pgp dans la membrane apicaleThe P-glycoprotein (Pgp), a drug efflux pump, is expressed in intestinal epithelial cells, where it constitutes a barrier against xenobiotic substrates. In inflammatory bowel disease, a dysregulation in the production of IL2, IFNg and TNFa, and an alteration of Pgp expression and activity have been reported. The aim of our study was to investigate the effects of each cytokine on Pgp activity, expression and localization in Caco-2 cells model. IL2 and TNFa induced both a diminution of MDR1 mRNA by semi-quantitative RT-PCR and a significant decrease of Pgp activity with a normal localisation of Pgp by confocal laser scanning microscopy. In contrast, IFNg induced up-regulation of both mRNA MDR1 and Pgp protein expression without incidence on Pgp activity but a delocalization of Pgp was observed in apical plasma membrane
32
Amamou, Asma. "Le récepteur minéralocorticoide : une cible potentielle dans la fibrose intestinale ? Mineralocorticoid receptor antagonisl improves inflammation and fibrosis in chronic DSS colitis mouse model Neutrophil gelatinas-associated lipocalin (NGAL) is a mineralocorticoid receptor target involved in intestinal inflammation and fibrosis Inflammatory bowel diseases and food additives : to add fuel on the flames Dietary salt activates intestinal fibroblasts, thereby contributing to exacerbation of intestinal fibrosis Dietary aryl hydrocarbon receptor ligands have no anti-fibrotic properties in transforming growth factor-β1-stimulated human colonic fibroblasts Effet d'un régime riche en sel sur la fibrose intestinale dans un modèle murin de colite chronique Etude de l'interaction entre des dérivés du tryptophane et le récepteur aryl hydrocarbone dans un modèle in vitro de fibrose intestinale." Thesis, Normandie, 2020. http://www.theses.fr/2020NORMR079.
Abstract:
Les maladies inflammatoires chroniques de l’intestin (MICI) se développent chez des individus génétiquement prédisposés sous l’influence de facteurs environnementaux. La fibrose intestinale est une complication fréquente de ces MICI sans traitement spécifique, qui se caractérise par une accumulation de matrice extracellulaire synthétisée par les cellules mésenchymateuses. Le récepteur minéralocorticoïde (RM) est l’effecteur terminal du système hormonal rénineangiotensine-aldostérone (SRAA). Le RM et l’ensemble des composants du SRAA sont exprimés dans le tractus gastro-intestinal et leur expression est augmentée dans l’intestin des patients atteints de MICI. L’antagonisme du RM exerce des effets bénéfiques sur la réduction de l’inflammation et de la fibrose extra-intestinales.L’objectif principal de ce travail de thèse était de déterminer si l’antagonisme du RM exerce des effets bénéfiques sur la fibrogenèse intestinale et d’en identifier les mécanismes sous-jacents. Un modèle de colite chronique induite chez la Souris et des modèles cellulaires de fibrose intestinale ont été utilisés. L’antagonisme du RM a été étudiée par des approches pharmacologiques et par invalidation génétique. Nous avons montré que l’inhibition pharmacologique ou génétique du RM diminue l’inflammation et la fibrose intestinales dans le modèle de colite chronique chez la Souris. L’activation du RM par l’aldostérone augmente la prolifération ainsi que l’expression de TGF-β1 des fibroblastes coliques humains et promeut la transition endothélio-mésanchymateuse, des cellules endothéliales vasculaires intestinales humaines. Nous avons également montré que la lipocaline associée à la gélatinase des neutrophiles (NGAL) est une cible du RM au niveau de l’intestin et est responsable des effets pro-fibrotiques médiés par l’activation du RM. L’invalidation génétique de la NGAL inhibe la voie de signalisation du TGF-β1 dépendante des SMAD, qui joue un rôle central dans l’initiation et le développement de la fibrose. En conclusion, nous avons d’une part démontré l’implication du RM dans la fibrose intestinale et d’autre part montré que ses effets étaient dépendants de la NGAL. Ainsi, l’antagonisme du RM pourrait constituer une nouvelle cible thérapeutique dans la fibrose intestinale associée aux MICI et permettrait de repositionner des molécules déjà disponibles dans le contexte des MICIInflammatory bowel diseases (IBD) occur in people with a genetic predisposition under the influence of environmental factors. Intestinal fibrosis is a common complication in IBD with no specific therapy which is characterized by an accumulative deposit of extra-cellular matrix produced by mesenchymal cells. Mineralocorticoid receptor (MR) is the final effector of renin-angiotensin-aldosterone system (RAAS). MR and all components of RAAS are expressed in the gastrointestinal tract and are up-regulated in the intestine from IBD patients. MR antagonism exerts beneficial properties in inflammation and fibrosis from extra-intestinal organs. We aimed to investigate whether MR antagonism had beneficial effects in intestinal fibrogenesis using murine chronic colitis and cellular models of intestinal fibrosis. MR antagonism was investigated by a dual approach using pharmacological inhibition and genetic invalidation. In the present study, we have demonstrated that pharmacological or genetic MR antagonism reduced inflammation and intestinal fibrosis in murine DSS chronic chemically-induced colitis. MR activation by aldosterone increased cell proliferation and TGF-β1 production in human colonic fibroblasts and human intestinal endothelial cells. Lipocalin associated with neutrophil gelatinase (NGAL) mediated pro-fibrotic effects via the activation of RM by aldosterone. Genetic invalidation of NGAL also reduced the SMAD-dependent TGF-β1 signaling pathway. In conclusion, we have demonstrated the MR involvement in intestinal fibrosis and these effects are mediated through NGAL. Thus, MR antagonism may represent a novel attractive approach in the treatment of intestinal fibrosis associated with IBD and may allow the repositioning molecules already available in the field of IBD
33
Kleta, Sylvia. "Einfluss des probiotischen Escherichia coli Nissle 1917 (EcN) auf die Infektion mit atypischen enteropathogenen E. coli (aEPEC) im porcinen in vitro-Modell." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15941.
Abstract:
In der vorliegenden Arbeit wurde in einem in vitro-Modell mit porcinen intestinalen Epithelzellen (IPEC-J2) der Einfluss des probiotischen E. coli Nissle 1917 (EcN) auf die Infektion mit atypischen EPEC (aEPEC) untersucht. EcN reduzierte bei Vorinkubation auf IPEC-J2 die aEPEC-Infektion drastisch. Konfokale Laserscanning- und Elektronenmikroskopie zeigten, dass EcN die Adhäsion und Mikrokoloniebildung inhibierte, jedoch nicht die Ausbildung von Attaching and Effacing-Läsionen adhärenter aEPEC. Der inhibierende Effekt von EcN wurde durch dessen sehr gute Adhäsionsfähigkeit an IPEC-J2 vermittelt. Die F1C-Fimbrien wurden als wichtigster Adhäsionsfaktor von EcN identifiziert. Darüber hinaus waren auch H1-Flagellen durch Ausbildung interbakterieller Verbindungen maßgeblich an der Adhäsion des Stammes beteiligt. In gleichem Maß wie die Vorinkubation von EcN reduzierte die Koinkubation seines Kulturüberstandes die aEPEC-Infektion, was auf die Abgabe eines inhibierenden Faktors in den Kulturüberstand schließen lässt. Dieser Faktor wurde auch von anderen pathogenen sowie nicht pathogenen E. coli-Stämmen in Schüttelkultur gebildet und scheint deshalb nicht spezifisch für EcN zu sein. Jedoch ermöglichte erst die gute Adhäsionsfähigkeit von EcN auf der Epithelzelloberfläche die Abgabe ausreichender Mengen des Inhibitors und eine Beeinflussung der aEPEC-Infektion. Die Ergebnisse weisen darauf hin, dass durch EcN die initiale Anheftung von aEPEC an die Wirtszelle unterbunden wird. Der inhibierende Effekt von EcN auf die aEPEC-Infektion war zeitabhängig. Im Gegensatz zur Vorinkubation erhöhten Ko- und Nachinkubation von EcN die Adhäsion von aEPEC und hatten einen geringeren inhibierenden Effekt auf die Mikrokoloniebildung. Dieser gegensätzliche Effekt auf die Adhäsion von aEPEC wird möglicherweise von einem zweiten Faktor hervorgerufen. Dieser scheint nur dann wirksam zu sein, wenn der inhibierende Faktor in zu geringer Konzentration oder erst nach Adhäsion von aEPEC vorliegt.In this study, the effects of the probiotic E. coli strain Nissle 1917 (EcN) on host cell infection with atypical enteropathogenic E. coli (aEPEC) were investigated in an in vitro porcine intestinal epithelial cell model (IPEC-J2). In pre-incubation experiments, EcN drastically reduced the infection efficiencies of aEPEC. Using confocal laser scanning microscopy and scanning electron microscopy, it was shown that EcN inhibited the attachment and formation of microcolonies, but not the formation of attaching and effacing lesions by adherent aEPEC. The inhibitory effect was mediated by the adherent properties of EcN to epithelial cells. The F1C fimbriae were identified as the most important adhesion factor of EcN in vitro. Furthermore, the H1 flagellae were also shown to be involved in the adhesion of EcN, serving as bridges between bacterial cells. Co-incubation of culture supernatants of EcN reduced the infection efficiencies of aEPEC to the same extent as in pre-incubation with EcN bacteria, indicating the secretion of an inhibitory factor by EcN. This factor was also secreted by other pathogenic and non-pathogenic E. coli strains in shaking culture and therefore does not appear to be specific for EcN. However, the outstanding ability of EcN to adhere to epithelial cells largely contributes to the secretion of sufficient concentrations of this inhibitory factor und to the influence on the aEPEC infection. The results suggest that EcN interferes with the initial adhesion of aEPEC to host cells. The inhibitory effect of EcN was found to be time-dependent. In contrast to pre-incubation experiments, co- and post-incubation of EcN actually increased the adhesion efficiencies of aEPEC and showed only minor effects on microcolony formation. This second effect of EcN on aEPEC adhesion, possibly due to a second factor, appears only to be effective when the putative inhibitory factor is either present at low concentrations or after aEPEC is already adherent to host cells.
34
Ménez-Berlioz, Cécile. "Intéractions de la miltefosine avec la barrière intestinale : étude des mécanismes de capture et de transport sur un modèle in vitro de cellules CACO-2 : application à la conception d'une bithérapie antileishmanienne." Paris 11, 2006. http://www.theses.fr/2006PA114834.
Abstract:
La miltéfosine (hexadecylphosphocholine, HePC) est une nouvelle molécule disponible pour le traitement de la leishmaniose viscérale. Elle a comme particularité et avantage majeur d'être active après une administration par voie orale. La voie orale est une voie d'administration de choix: elle est non invasive, souvent de faible coût et favorise l'observance du traitement. Le passage de la miltéfosine à travers la barrière intestinale est très peu décrit dans la littérature. Ce travail de thèse a donc comme objectif d'étudier les effets de la miltéfosine sur l'épithélium intestinal et de déterminer les mécanismes mis en jeu lors de sa capture et de son transport sur un modèle in vitro composé de cellules épithéliales Caco-2 en culture. Ces données ont été exploitées pour développer une bithérapie antileishmanienne administrée par voie orale: la potentialité de la miltéfosine à améliorer la biodisponibilité orale de l'amphotéricine B, un autre agent antileishmanien, a été évaluéeMiltefosine (hexadecylphosphocholine, HePC) has recently been developed for the treatment of visceral leishmaniasis. It has the particular advantage of being active after oral administration. The oral route is preferable because it is non invasive, is often less expensive and favours compliance with treatment. There is not much information in the literature about the passage of miltefosine across the intestinal barrier. Therefore, the aims of this thesis were to study the effects of miltefosine on the intestinal epithelium and to determine the mechanisms involved in its uptake and transport, using a model of Caco-2 cells in culture. The results were applied to the development of antileishmanial bitherapy by the oral route: the ability of miltefosine to improve the oral bioavailability of another antileishmanial agent, amphotericin B, was evaluated
35
Tria, Scherrine. "Novel in vitro models for pathogen detection based on organic transistors integrated with living cells." Phd thesis, Ecole Nationale Supérieure des Mines de Saint-Etienne, 2013. http://tel.archives-ouvertes.fr/tel-00972057.
Abstract:
In biological systems, different tissues have evolved to form a barrier. An example is the intestinal epithelium, consisting of a single layer of cells lining the wall of the stomach and colon. It restricts the passage of harmful chemicals or pathogens from the light into the tissue, while selectively absorbing the most nutrients, electrolytes and water are necessary for the host. Tight junctions are structures which limit the passage of the material through the space between the cells. The ability to measure the paracellular and transcellular transport is of vital importance because it provides a wealth of information on the state of the barrier, indicative of certain disease states , since the disruption or malfunction of the structures involved in the transport through the tissue barrier is often caused or is indicative of toxicity or disease. In addition, the degree of integrity of the barrier is a key indicator of the relevance of a particular model in vitro for use in toxicology and drug screening. The advent of organic electronics has created a unique opportunity to connect the worlds of electronics and biology, using devices such as organic electrochemical transistor (OECT), which provides a very sensitive way to detect ionic currents. These devices have unprecedented sensitivity in a format that can be mass produced at low cost.The purpose of this study was to integrate a monolayer of cells representative of the gastro intestinal barrier with OECTs , to create devices that detect disruptions of the barrier in a timely and sensitive manner. This technique was demonstrated to be at least as sensitive, but a higher speed than current techniques on the market
36
Domenech, Cabrera Josefa. "Bioavailability and effects of microplastics and nanoplastics on in vitro gastrointestinal models. A focus on nanopolystyrene as representative nano-sized plastic material." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/674024.
Abstract:
Els plàstic s’utilitzen en incomptables aplicacions en diferents àmbits, com ara la construcció, l’agricultura, o la medicina, degut a la facilitat amb que es poden modificar les seues propietats fisicoquímiques, i la seua producció ràpida i de baix cost. És per això que la generació de residus plàstics que acaba contaminant el medi ambient creix de manera exponencial. Diferents factors ambientals afavoreixen processos químics, físics i biològics que fan que aquests residus es degraden, generant els micro- i nanoplàstics (MNPLs). A més a més, la nanotecnologia genera una gran quantitat de partícules de plàstic de mides nano- i micromètriques que s’utilitzen en cosmètics o productes de neteja, i acaben contribuint a la contaminació ambiental per plàstics.
Aquest repte mediambiental va lligat a la exposició humana. No obstant, les actuals limitacions metodològiques no permeten fer estimacions precises sobre els nivells d’exposició humana als MNPLs, i la manca de dades sobre els efectes d’aquests en models de mamífer fa difícil establir si els MNPLs suposen un risc per a la salut humana. Amb la finalitat de contribuir al coneixement sobre l’exposició humana als MNPLs i els seus possibles efectes tòxics i genotòxics, hem dut a terme una revisió bibliogràfica de les últimes publicacions sobre la distribució dels MNPLs en aliments i l’aire que respirem, i hem fet estudis en models in vitro per esclarir els efectes biològics de les nanopartícules de poliestirè (PSNPs).
La revisió bibliogràfica, a més d’assenyalar les limitacions i els aspectes menys estudiats que condicionen l’avaluació de riscos dels MNPLs, defineix la ingestió com la major via d’exposició humana als MNPLs. Per això, hem estudiat les interacciones de les PSNPs amb diferents models in vitro de l’intestí humà, així com la toxicitat i la genotoxicitat d’aquests nanoplàstics en els nostres models. El nostre primer estudi confirma la capacitat d’internalització de les PSNPs en les cèl·lules Caco-2. No obstant, malgrat que les PSNPs arriben als nuclis cel·lulars en només 24 h, no produeixen efectes genotòxics.
Degut a l’interès en els possibles efectes que els MNPLs poden produir un cop ingerits, hem analitzat la internalització de les PSNPs en models in vitro que imiten la barrera intestinal humana, que inclouen la representació de les cèl·lules caliciformes (Caco-2/HT29) i les cèl·lules M (Caco-2/HT29/Raji-B). Els resultats obtinguts assenyalen les PSNPs com agents poc tòxics degut a la poca capacitat que tenen d’induir ROS o altres efectes. A més, malgrat la facilitat amb la que les PSNPs internalitzen, arriben als nuclis i travessen les barreres, no es van trobar efectes genotòxics o dany oxidatiu a l’ADN de les cèl·lules que formen aquests models.
La potencial capacitat dels MNPLs per actuar com a vectors d’altres contaminants tòxics fa més greu l’escenari d’exposició. Per això, hem introduït el disseny de co-exposició amb l’objectiu de cobrir aquest aspecte. Els nostres resultats demostren la interacció física entre les PSNPs i les nanopartícules de plata (AgNPs), utilitzades com a model de contaminant ambiental estès de manera global. Amb aquest estudi, hem confirmat la interacció física entre metalls/MNPLs utilitzant metodologies clàssiques adaptades als reptes actuals. A més, hem observat l’increment de la internalització de les AgNPs quan aquestes es combinen amb les PSNPs, malgrat que aquesta co-exposició no va induir efectes tòxics. D’una altra banda, es va demostrar que els complexos AgNPs/PSNPs internalitzen en les cèl·lules Caco-2 i arriben als nuclis cel·lulars, causant un increment en la tendència al dany genotòxic conforme augmenta la concentració de AgNPs quan es combina amb dosis altes de PSNPs. El nitrat de plata es va utilitzar en paral·lel en aquest estudi com a agent alliberador de ions de plata.Hoy en día los plásticos se usan en incontables aplicaciones en diferentes ámbitos, como la agricultura, la medicina, la construcción o la electrónica, debido a la facilidad con que se puede modificar sus propiedades fisicoquímicas, y su producción rápida y de bajo coste. Esto ha dinamitado la generación de residuos plásticos que acaba contaminando el medioambiente. Distintos factores ambientales favorecen procesos químicos, físicos y biológicos que hacen que la basura plástica se degrade, generando los micro- y nanoplásticos (MNPLs). Además de estos, la nanotecnología genera una gran cantidad de partículas de plástico en las escalas de tamaño micro- y nano, que se usan en cosméticos o productos de limpieza, y acaban contribuyendo a la contaminación ambiental por plásticos. Este reto medioambiental está acompañado por la exposición humana. Sin embargo, las actuales limitaciones metodológicas no permiten hacer estimaciones precisas sobre el nivel de exposición humana a los MNPLs, y la escasez de datos sobre los efectos de los MNPLs en modelos de mamífero hacen que sea difícil definir si los MNPLs suponen un riesgo para la salud humana. Con el fin de contribuir al conocimiento sobre la exposición human a los MNPLs y sus posibles efectos tóxicos y genotóxicos, hemos llevado a cabo una revisión de las últimas publicaciones acerca de la distribución de los MNPLs en los alimentos y el aire que respiramos, y hemos realizado estudios en modelos in vitro para esclarecer los efectos biológicos de las nanopartículas de poliestireno (PSNPs). La revisión bibliográfica, además de señalar las limitaciones y lagunas de conocimiento que condicionan la evaluación de riesgos de los MNPLs, reveló que la ingestión es la mayor vía de exposición humana a los MNPLs. Por eso, estudiamos las interacciones de las PSNPs con diferentes modelos in vitro del intestino humano, así como la toxicidad y la genotoxicidad de los nanoplásticos en estos modelos. Con el primer estudio, confirmamos la capacidad de internalización en células Caco-2 de las PSNPs. Sin embargo, a pesar de que las PSNPs alcanzan el núcleo de las células en tan sólo 24 h, no producen efectos genotóxicos. Debido al creciente interés en los posibles efectos que pueden producir los MNPLs tras su ingestión, analizamos la internalización de las PSNPs en modelos in vitro que imitan la barrera intestinal humana, incluyendo la representación de las células caliciformes (Caco-2/HT29) y las células M (Caco-2/HT29/Raji-B). Nuestros resultados apuntan a las PSNPs como agentes poco tóxicos debido a su poca capacidad para inducir ROS o otros efectos. Además, a pesar de la facilidad con la que las PSNPs internalizaron, alcanzaron el núcleo e incluso atravesaron las barreras, no produjeron genotoxicidad o daño oxidativo en el ADN de las células que forman dichos modelos. La potencial capacidad de los MNPLs para actuar como vectores de otros contaminantes tóxicos agrava el escenario de exposición. Por eso, hemos introducido un diseño de co-exposición con el objetivo de cubrir este aspecto. Nuestros resultados demostraron la interacción física entre las PSNPs y las nanopartículas de plata (AgNPs), usadas como modelo de contaminante ambiental extendido globalmente. Con este estudio, confirmamos la interacción física entre metales/MNPLs usando metodologías clásicas adaptadas a los retos actuales. Además, observamos un incremento en la internalización de las AgNPs cuando éstas fueron combinadas con PSNPs, aunque esta co-exposición no indujo efectos tóxicos. Por otro lado, demostramos que los complejos AgNPs/PSNPs internalizan en las células Caco-2 y alcanzan el núcleo celular, causando un aumento en la tendencia al daño genotóxico conforme aumenta la concentración de AgNPs cuando se combina con dosis altas de PSNPs. El nitrato de plata se introdujo paralelamente en este estudio como agente liberador de iones de plata.
Owing to the wide range of tuneable physicochemical properties, and the rapid and low-cost production, plastics present uncountable applications in industry such as product packaging, agriculture, medical applications, building or electronics, among others. This fact leads to the corresponding exponential increase in the generated plastic waste that ends up contaminating the environment. Different environmental conditions favour physical, chemical and biological processes that drive plastic waste to a continuous degradation, generating the so-called micro- and nanoplastics (MNPLs). In addition to those, rapid advances in nanotechnology have driven to the target industrial production of plastic particles in the micro- and nano-size ranges used in cosmetic or cleaning products that contribute to plastic environmental pollution. The increasing presence of MNPLs in nature represents an environmental challenge but this is also accompanied by the human exposure. Nevertheless, the current methodological limitations do not allow for accurate estimations on the levels of human exposure, and the scarcity of data on the effects of MNPLs in mammalian models hampers the understanding of whether the presence of MNPLs in different environmental niches and organisms may pose a risk for humans. Aiming to contribute to expand the knowledge on human exposure and the potential toxic and genotoxic effects of MNPLs on human health, we have reviewed the last publications referring to the occurrence of MNPLs in food and airborne, and we have performed extended in vitro studies on the biological effects of polystyrene nanoparticles (PSNPs). Our review of literature revealed ingestion as the major human exposure route to MNPLs and highlighted the knowledge gaps and limitations conditioning the MNPLs hazard assessment. Therefore, we moved forward to evaluate the interactions of PSNPs with different human gastrointestinal in vitro models, as well as toxicity and genotoxicity of the nanoplastics in those models. We confirmed the ability of PSNPs to internalise into human-derived undifferentiated Caco-2 cells. Importantly, Caco-2 cells showed signals of general stress induction after the exposure to PSNPs. However, although PSNPs reached the cell nuclei in only 24 h, cells did not show genotoxicity. Because of the heightened interest in the effects of MNPLs after their ingestion, we further analysed, the internalisation ability of PSNPs in Caco-2 based in vitro 2D models of the gut barrier which include representation of goblet (Caco-2/HT29) and microfold-cells (Caco-2/HT29/Raji-B). Our findings describe PSNPs as weak toxicants due to their low ability to induce ROS or other toxic effects. Similar to that found in the undifferentiated Caco-2 model, PSNPs did not exert genotoxic or oxidative DNA damage despite having a great internalisation capacity which allowed reaching cell nuclei and translocation across the barriers. Although literature regarding MNPLs health effects show controversy, the exposure scenario is aggravated if the MNPLs’ ability as carriers of other hazard contaminants is considered. Therefore, we attempted to cover this aspect by introducing the co-exposure design. Our results demonstrated the physical interaction between PSNPs and a widespread legacy pollutant, silver nanoparticles (AgNPs). Importantly, we proved the adaptability of classical methodological approaches to novel technical challenges as it is the visualisation of the metals/MNPLs interplay. In addition, we observed an enhancement of AgNPs internalisation by undifferentiated Caco-2 cells, after the co-exposure to AgNPs/PSNPs, but not a higher induction of toxic effects after the co-treatment. On the other hand, AgNPs/PSNPs complexes internalised and reached Caco-2 cell nuclei, causing an increasing tendency to genotoxic damage as the AgNPs concentration increases when combined with high doses of PSNPs. Silver nitrate (AgNO3) was included in this study as a surrogate of silver ion releasing agent to confirm that the effects induced by AgNPs were not caused by released ions.
Universitat Autònoma de Barcelona. Programa de Doctorat en Genètica
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Benkhelifa, Samir. "Evaluation des mécanismes d'absorption intestinale des céphalosporines a-aminées : utilisation de modèles "in vitro" et "ex vivo"." Paris 5, 1995. http://www.theses.fr/1995PA05P620.
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Clarke, Sarah Louise. "Analysis of murine intestinal intraepithelial lymphocytes in vitro." Thesis, King's College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412544.
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Patient, J. D. "Development of an in-vitro epithelial-myofibroblast intestinal model." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33617/.
Abstract:
In vitro studies of drug permeability are traditionally carried out using cultured monolayers of epithelial cell lines grown on semi permeable membranes. Caco-2 cells, which are colon-derived, spontaneously form polarised cell layers when cultured in vitro which are akin to an epithelium of enterocyte-like cells. The Caco-2 model has been developed as a powerful in vitro tool in the early assessment of human drug permeability and is even approved by regulatory agencies for biowaver applications (i.e. in vitro tests in lieu of in vivo animal experiments). As Caco-2 cells are derived from colon tissue they represent a more formidable barrier to drug absorption than the upper regions of the intestine which is where the majority of oral drugs permeate into the body. Whilst the Caco-2 model, alongside other in vitro methods, has provided a significant means to understand the mechanics of drug permeability. Many researchers have sought to improve upon the existing unicellular model; it is hoped that this will result in a more relevant and predictive model for researchers to test new drugs but also to dissect cellular cross talk and to probe cell-matrix interactions. Myofibroblasts are a niche cell type located subjacent to epithelial tissues which regulate the integrity, growth and differentiation of the overlying epithelium. In this study the co-culture of human epithelial cell lines with a myofibroblast cell line, CCD-18co, were investigated to study how myofibroblasts influence the barrier integrity of epithelia in vivo. Additionally, nanofibre scaffolds produced by electrospinning were explored as 3-dimensional and topographically relevant cell scaffolds to support the growth of intestinal cells in vitro. In a traditional transwell format, cultured epithelial lines Caco-2 (intestinal), HT-29 (intestinal) and Calu-3 (airway) in co-cultures with CCD-18co revealed cell-line specific response with respect to the modulation of barrier integrity. The mechanism of the modulation was confirmed to be mediated through paracrine signalling by using myofibroblast conditioned media. Fibre scaffolds which mimic the fibrillar nature of the extra cellular matrix and basement membrane were produced by electrospinning using the polymer, poly (ethylene terephthalate). Nanofibre scaffolds were characterised and further optimised for cell culture with surface coating with collagen to achieve adequate cell attachment and confluence. Work was also conducted to incorporate villi architectures into the fibre scaffolds; the potential of this ambition was investigated by using models produced by rapid prototyping. These models, which demonstrated good fidelity with the actual villi dimensions found in vivo, were used during the electrospinning process to shape the polymer scaffolds towards the geometry found in intestinal tissue. A number of molecular tracers and model drug compounds were used to evaluate the permeability profiles of Caco-2 monocultures, Caco-2/CCD-18co co-cultures cultured on the two different culture substrates in addition to the assessment of resected porcine intestinal tissue sections. Caco-2 cells and Caco-2/CCD-18co co-cultures grown on nanofibre substrates were found to have lower electrical resistance and higher permeability properties than their transwell equivalents. Caco-2/CCD-18co co-cultures on conventional transwell inserts demonstrated a permeability profile closer to the resected porcine tissue and reported human tissue values than the conventional Caco-2 model whilst maintaining p-glycoprotein assay sensitivity. This work forms a solid foundation for further research into the role of myofibroblasts in epithelial cell function and in the development of more predictive in vitro cell models for widespread scientific research.
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Alhamoruni, Abdissalam Ag Ali. "In vitro studies of huam intestinal permeability and modulation." Thesis, University of Nottingham, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.537654.
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Fordham, Robert Peter. "Maturation of intestinal epithelial progenitors, in vitro and in vivo." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648458.
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Accoceberry, Isabelle. "Microsporidiose humaine intestinale a enterocytozoon bieneusi modeles in vivo et in vitro - purification des spores du parasite." Paris 6, 2001. http://www.theses.fr/2001PA066002.
Abstract:
Enterocytozoon bieneusi, parasite de decouverte recente, responsable de diarrhees chroniques chez les patients infectes par le virus de l'immunodeficience humaine (vih) et tres immunodeprimes a suscite un effort de recherche particulier. Notre travail a fait l'objet de trois approches : 1- pour l'etude in vivo, nous avons d'abord experimente differentes especes animales immunodeprimees par corticotherapie. Les lapins hybrides new-zealand/cr et surtout les rats adultes sprague-dawley sont les plus sensibles a l'infection qui a ete etablie chez 100% des animaux et a persiste jusqu'a leur mort ou leur sacrifice 40 a 50 jours apres l'inoculation par voie orale. Pour la premiere fois la possibilite d'obtenir une infection a e. Bieneusi d'origine humaine chez un animal de laboratoire a ete etablie. Cependant, malgre les tentatives d'optimisation, l'infection induite est restee chronique, asymptomatique avec une faible excretion de spores dans les selles et sans alterations notables au niveau de l'epithelium intestinal. Dans un second temps, l'infestation d'animaux congenitalement immunodeficients, souris rag-2 ne produisant pas de lymphocytes t et b matures et souris ifn- r 0 / 0 n'exprimant pas le gene codant pour le recepteur a l'ifn-, s'est traduit par une infection plus faible que chez les rats sprague-dawley. Ce resultat suggere le role probable des cellules nk, des lymphocytes tcd8+ cytotoxiques et de l'ifn dans le controle de l'infection. Les essais d'isolement d'e. Bieneusi in vitro sur differents types de lignees cellulaires ont ete rapidement limites par les contaminations bacteriennes et/ou fongiques. Les difficultes rencontrees par toutes les equipes pour obtenir un modele exploitable d'infection a e. Bieneusi nous
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Tomlinson, Sophie. "Development of an in vivo-like in vitro intestinal epithelial model." Thesis, University of Nottingham, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.523450.
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Eastwood, Martin. "An In Vitro Approach to Assess Intestinal Drug Metabolism and Transport." Thesis, University of Manchester, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493464.
Abstract:
The epithelium of the small intestine provides a physical and biochemical banner that restricts the transcellular absorption of orally dosed drugs into the bloodstream through mechanisms including P-glycoprotein (Pgp) and Cytochrome P450 3A4 (CYP3A4). P-glycoprotein is constitutively expressed on the apical membrane of the intestinal lumen where it can act to limit xenobiotic absorption by excreting drugs that have reached the cell interior back into the intestinal lumen. CYP3A4, the most abundant phase one drug metabolising enzyme in humans, can metabolise over fifty percent of commercially available drugs and is located apically within the enterocytes where it can restrict bioavailability by metabolising xenobiotics.
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Black, Annie. "Angiogénèse in vitro dans un modèle d'équivalent dermo-épidermique humain." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/mq26166.pdf.
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Winsor, Geoffrey L. "The intestinal epithelial cell as a central component of the intestinal cytokine network, an in vitro model of chemokine production." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0017/MQ57337.pdf.
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Bajka, Balazs Hendrik. "The characterization of an in vitro model of small intestinal permeability dysfunction /." Title page and abstract only, 1998. http://web4.library.adelaide.edu.au/theses/09SB/09sbb165.pdf.
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Fitzhenry, Robert James. "Interactions of enteropathogenic and enterohaemorrhagic escherichia coli with intestinal mucosae in vitro." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407237.
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Baxter, P. S. "An in vitro and in vitro study by electrical methods of small intestinal ion transport in cystic fibrosis." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596483.
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Betts, Andrea M. "An investigation into the intestinal absorption of melphalan in vivo and in vitro." Thesis, University of Aberdeen, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235651.
Abstract:
Melphalan (the synthetic product of nitrogen mustard and L-phenylalanine) is an alkylating agent and is the drug of choice in the treatment of multiple myeloma. The bioavailability of melphalan is variable but factors affecting its absorption and the mechanism(s) by which the drug crosses the intestinal epithelium are not known. A sensitive assay for melphalan in plasma (down to a concentration of 2ng.ml-1) has been developed. The method, involving solid phase extraction and derivatisation with o-phthalaldehyde followed by reversed-phase high-performance liquid chromatography, was applied to the study of melphalan pharmacokinetics in multiple myeloma patients. Bioavailability ranged from 0.16 to 1.37 in nine fasting patients and peak plasma concentrations of melphalan ranged from 8.0 to 170 ng.ml-1 and occurred 0.5 to 2.0 hours after an oral dose of 10mg. When melphalan was taken with food (three patients) a mean reduction of 40% in bioavailability was observed. Significant correlations (P< 0.05) were observed between bioavailability and melphalan plasma concentrations in single samples drawn at 0.5, 1.0 and 2.0 hours. There was no significant correlation between renal function and melphalan absorption, distribution or elimination. A second peak was observed in the distribution phase of six of the eleven plasma concentration-time curves when melphalan was administered intravenously. The secondary peak may be attributed to either melphalan redistribution or enterohepatic circulation. An in vitro method (modified Ussing technique) was developed to investigate melphalan transport across rat and human small intestine. There was no evidence for the Na+-coupled (active) transport of melphalan in these tissues. The rate of melphalan transfer was non-saturable and values of apparent mass transfer coefficients were comparable with the diffusional contribution to the intestinal transport of amino acids The results indicate that passive diffusion is the major process responsible for the transfer of melphalan across intestinal epithelium.