Les bénéfices attribués aux probiotiques sont nombreux : effets bénéfiques sur le microbiote intestinal, confort digestif, modulation du système immunitaire et prévention des infections intestinales. Ces effets divers et variés sont souches-spécifiques. Actuellement, de nombreuses études visent à mieux caractériser ces effets probiotiques.Ces travaux avaient pour objectif de déterminer et d’analyser les effets probiotiques de trois souches de la collection Danone : la souche de Lactobacillus paracasei CNCM I-3689, la souche de L. rhamnosus CNCM I-3690 ainsi que la souche de Bifidobacterium animalis subps. lactis CNCM I-2494. Dans un premier temps, leurs effets ont été caractérisés dans des modèles in vitro pour des propriétés anti-pathogènes, des propriétés d’immuno-modulation ainsi que pour des propriétés de protection de la barrière épithéliale intestinale. Dans un second temps, leurs effets de la protection de la barrière ont été confirmés dans un modèle murin de faible inflammation.Une analyse des mécanismes sous-jacents à ces effets à la fois chez la souche probiotique et chez l’hôte a été initiée par la construction et l’analyse fonctionnelle de banques génomiques bactériennes ainsi que par l’étude de la modulation des gènes impliqués dans le maintien de la barrière intestinale chez l’hôte. / Health benefits of probiotic bacteria are numerous: beneficial effects on the intestinal microbiota, digestive comfort, modulation of the immune system and prevention of winter infection. These diverse and various effects are strain-specific. Nowadays, numerous studies aim at better characterizing those probiotics effects.This project aimed at identifying and analyzing the probiotic effects of three strains from Danone collection: Lactobacillus paracasei CNCM I-3689, L. rhamnosus CNCM I-3690 and Bifidobacterium animalis subsp. lactis CNCM I-2494. First, their effects were assessed in in vitro models for immunomodulation properties, antipathogens activity and intestinal barrier protection. Secondly, their beneficial effects were confirmed in low-grade inflammation murine model.The analysis of the underlying mechanisms has been initiated both in the bacterial strains by the construction and the functional analysis of genomic libraries and in the host by measuring the modulation of the genes involved in the intestinal barrier maintain.

<p>One of the main obstacles to maintaining patients with short bowel syndrome on parenteral nutrition, or successfully transplanting these patients with a small bowel graft, is the many severe infections that occur. Evidence is accumulating that translocating bacteria from the patient’s bowel causes a significant part of these infections. In this thesis bacterial translocation is studied in a Thiry-Vella loop of defunctionalised small bowel in the rat. </p><p>Bacterial translocation to the mesenteric lymph nodes (MLNs) occurs in almost 100% of the rats after three days. No systemic spread of bacteria is observed unless there is additional immunosupression with depletion of Kupffer cells in the liver. However, blocking the function of α/β T cells does not increase the translocation. Removal of MLNs does not either aggravate bacterial translocation in the Thiry-Vella loop model. Conversely, after small bowel transplantation translocating bacteria spread systemically if the MLNs are removed. </p><p>The Thiry-Vella loop should also be a suitable model for the testing of potentially translocation-inhibiting substances. Reinforcement of the intestinal barrier with glutamine or phosphatidylcholine proved insufficient in decreasing bacterial translocation. Even selective bowel decontamination with tobramycin failed to abolish bacterial translocation. Thus, it seems that the driving force for translocation in this model is strong regardless of the relatively small trauma of intestinal defunctionalisation.</p><p>Flow cytometric studies of the immune cells in the spleen MLNs showed a decrease in MHC class II positive T cells in the MLNs of the Thiry-Vella loop. Concurrently the number of macrophages increased with time as observed by immunohistochemistry. The fraction of MHC class II negative macrophages increased in the spleens of rats treated with glutamine. </p><p>In conclusion, the Thiry-Vella loop model offers possibilities of immunological as well as mechanistic studies on bacterial translocation from small intestine.</p>

One of the main obstacles to maintaining patients with short bowel syndrome on parenteral nutrition, or successfully transplanting these patients with a small bowel graft, is the many severe infections that occur. Evidence is accumulating that translocating bacteria from the patient’s bowel causes a significant part of these infections. In this thesis bacterial translocation is studied in a Thiry-Vella loop of defunctionalised small bowel in the rat. Bacterial translocation to the mesenteric lymph nodes (MLNs) occurs in almost 100% of the rats after three days. No systemic spread of bacteria is observed unless there is additional immunosupression with depletion of Kupffer cells in the liver. However, blocking the function of α/β T cells does not increase the translocation. Removal of MLNs does not either aggravate bacterial translocation in the Thiry-Vella loop model. Conversely, after small bowel transplantation translocating bacteria spread systemically if the MLNs are removed. The Thiry-Vella loop should also be a suitable model for the testing of potentially translocation-inhibiting substances. Reinforcement of the intestinal barrier with glutamine or phosphatidylcholine proved insufficient in decreasing bacterial translocation. Even selective bowel decontamination with tobramycin failed to abolish bacterial translocation. Thus, it seems that the driving force for translocation in this model is strong regardless of the relatively small trauma of intestinal defunctionalisation. Flow cytometric studies of the immune cells in the spleen MLNs showed a decrease in MHC class II positive T cells in the MLNs of the Thiry-Vella loop. Concurrently the number of macrophages increased with time as observed by immunohistochemistry. The fraction of MHC class II negative macrophages increased in the spleens of rats treated with glutamine. In conclusion, the Thiry-Vella loop model offers possibilities of immunological as well as mechanistic studies on bacterial translocation from small intestine.

Dans de nombreuses pathologies digestives dont les maladies inflammatoires chroniques intestinales (MICI), l'intégrité de la barrière épithéliale est rompue. Cette perte d'intégrité est notamment due à la réduction ou la perte d'expression des jonctions adhérentes composées du complexe E-cadhérine/caténines. Il est donc important d'identifier de nouvelles molécules capables de réguler ce complexe dans les MICI. C'est dans ce contexte que nous nous sommes intéréssés à une levure non pathogène, Saccharomyces boulardii (Sb) utilisée dans la prévention et le traitement de désordres gastro-intestinaux et qui présente des bénéfices thérapeutiques chez les patients atteints de MICI, notamment en régulant l'intégrité de la barrière intestinale. L'étude que nous avons réalisée sur des explants tissulaires provenant de patients atteints de MICI nous a permis de mettre en évidence que le surnageant de Sb (Sbs) protège la morphologie tissulaire et maintient l'expression de la E-cadhérine à la membrane. In vitro nous avons également pu montrer que Sbs accélère la ré-expression de la E-cadhérine à la membrane en régulant son recyclage par les endosomes (Rab11A), entrainant ainsi la restauration et le renforcement de la barrière épithéliale intestinale. / Some intestinal pathologies including inflammatory bowel disease (IBD) are associated with an altered barrier function. The reduction or the lost of adherens junctions composed by E-cadherin/catenins complex are linked to changes in the barrier integrity. Characterization of molecules targeting the E-cadherin/catenins complex during IBD is crucial for the development of alternative therapies. From this perspective, we focus ours studies on a non pathogenic yeast, Saccharomyces boulardii, used to prevent and treat gastro-intestinal disorders and may have beneficial effects in IBD treatment, including the regulation of barrier integrity. Ours studies on colonic explants from IBD patients showed that Sb supernatant (Sbs) protects epithelial morphology and maintains E-cadherin expression at the cell surface. In vitro study pointed out that Sbs accelerated the recovery of E-cadherin at the cell membrane. This process involved the modulation of the recycling of E-cadherin by endosomes (Rab11A), leading to restoration and strengthening of intestinal barrier function.

<p>Vitamin E is a natural lipophilic antioxidant contains eight structurally related forms, i.e., α-, β-, γ-, δ-tocopherols (αT, βT, γT, and δT) and corresponding tocotrienols. Recent research indicates that vitamin E forms are differentially metabolized to various carboxychromanols. Some these vitamin E metabolites have been shown to exhibit strong anti-inflammatory and anticancer effects, yet little is known about their bioavailability. Without this knowledge, it is impossible to assess the role of vitamin E metabolism in biological functions of vitamin E forms and their protective effects on chronic diseases. While αT and γT appear to improved gut health, the underlying mechanisms are not well understood. Furthermore, specific forms of vitamin E such as γT have been reported to have cancer-preventing effects, but their anticancer efficacy is relatively modest. For these reasons, this dissertation focused on the characterization of the pharmacokinetic formation of vitamin E metabolites after supplementation, and the investigation of the underlying mechanisms of the protective effect of vitamin E forms, αT and γT, on gut health, as well as anticancer efficacy of the combination of aspirin and γT on carcinogen-induced colon tumorigenesis.</p><p><br></p><p>The first project focuses on characterizing the pharmacokinetic formation of vitamin E metabolites after single dose supplementation of γ-tocopherol-rich mixed tocopherol (γTmT) and δ-tocotrienol (δTE). With our recently developed LC/MS/MS assay for quantifying vitamin E metabolites, we can simultaneously quantify the level of short-chain, long-chain, and sulfated carboxychromanols in plasma, urine, and fecal samples of supplemented animals. In this study, we investigated the pharmacokinetics including excretion of vitamin E forms and the formation of their metabolites after a single dose intragastric administration of tocopherols and tocotrienols in rats. We also measured vitamin E metabolites in the serum obtained from healthy humans after gT supplementation. In the plasma of rat, the pharmacokinetic profiles of γT and δTE are described as the following: γT, Cmax = 25.6 ± 9.1 μM, Tmax = 4 h; δTE, Cmax = 16.0 ± 2.3 μM, Tmax = 2 h. Sulfated CEHCs and sulfated 11’-COOHs were the predominant metabolites in the plasma of rat with Cmax of 0.4-0.5 μM (Tmax ~ 5-7 h) or ~0.3 μM (Tmax at 4.7 h), respectively. In 24-h urine, 2.7% of γT and 0.7% of dTE were excreted as conjugated CEHCs, the major identified urinary metabolites. In the feces, 17-45% of supplemented vitamers were excreted as un-metabolized forms and 4.9-9.2% as metabolites. The majority of metabolites excreted in feces were unconjugated carboxychromanols, among which 13’-COOHs constituted ~50% of total metabolites. Interestingly, 13’-COOHs derived from δTE were 2-fold higher than 13’-COOH from γT. Unlike rats, γ-CEHC is the predominant metabolites found in human plasma, although 11’-COOHs and 13’-COOHs (sulfated and unconjugated) were elevated by >20 folds responding to γT supplement. In this study, we found that tocopherols and tocotrienols, when taken as supplements, are mainly excreted as un-metabolized forms and long-chain carboxychromanols in feces. High fecal availability of 13’-COOHs may contribute to modulating effects on gut health.</p><p><br></p><p>The second project of my dissertation investigated the effect of vitamin E forms, αT and γT, on intestinal barrier function in a cellular model and a mouse colitis model. Inflammatory bowel diseases (IBD) are chronic idiopathic inflammatory conditions characterized by disruption of intestinal barrier integrity. Previous studies by others and us had demonstrated that vitamin E forms, αT and γT, can protect against chemical-induced colitis in animal models. However, the role of these vitamin E forms on intestinal barrier function has not been studied. Herein, we investigated the potential protective effects of vitamin E forms, αT and γT, on intestinal barrier function in a Caco-2 colon epithelial cell model and a dextran sodium sulfate (DSS)-induced colitis mouse model. In Caco-2 cells, pretreatment with 25mM αT and γT attenuated Caco-2 monolayer barrier dysfunction induced by 10 ng/mL TNF-α/IFN-γ, suggesting that these vitamin E forms protect intestinal barrier integrity in this cellular model. In male BALB/c mice, the supplementation of αT (0.05%) or γTmT (0.05%) when given 3 weeks before DSS treatment or at the same time as DSS treatment alleviated DSS-induced fecal bleeding and diarrhea symptoms in mice, and attenuated colon inflammation and colitis-associated damages. Additionally, αT and γTmT supplementation attenuated DSS-induced intestinal barrier dysfunction, as indicated by improving the level of occludin, a tight junction protein, in the colon and reducing lipopolysaccharide-binding protein (LBP) in the plasma. Furthermore, gut microbiota analysis demonstrated that αT and γTmT supplementation could modulate intestinal microbiome composition in mice with DSS treatment. DSS treatment reduced the relative abundance of Lachnospiraceae compared to healthy mice, and supplementation of αT and γT partially reversed this effect. Interestingly, the family Lachnospiraceae has been reported to decrease in IBD patients. Our study demonstrated the protective effects of vitamin E forms on intestinal barrier integrity in a cell-based model and a colitis model in mice. Furthermore, we demonstrated that these vitamin E forms caused favorable changes in the intestinal microbial population under colitis condition.</p><p><br></p><p>The third project of my dissertation evaluated the anticancer efficacy of the combination of aspirin and γT using an azoxymethane (AOM)-induced and colitis-promoted colon tumorigenesis mouse model. Extensive inflammation in the colon promotes the development of colorectal cancer (CRC). Eicosanoid production by pro-inflammatory enzymes, cyclooxygenases (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) play a critical role in the initiation, progression, and invasion of CRC. Thus, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, have been recommended for chemoprevention of CRC. However, long-term use of aspirin can cause many side effects, and the anticancer activity of aspirin is very modest. Previously, we have demonstrated that the combination of γT with aspirin prolonged the anti-inflammatory activity of aspirin and alleviated aspirin-associated adverse effects in a carrageenan-induced inflammation model in rats. Additionally, we found that the combination of γT and aspirin has stronger anticancer activity than aspirin or γT alone against HCT-116 human colorectal carcinoma cells. Therefore, we examined the anticancer effect of the combination of 0.025% aspirin and 0.05% γT against AOM-induced and DSS-promoted tumorigenesis in mice. In this study, we have found that the combination of aspirin and γT, but not aspirin or γT alone, suppressed colon tumorigenesis in mice, as indicated by 40% and 50% reduction in the multiplicity of total polyps (P < 0.05) and large adenomatous polyps (>2mm2, P < 0.05), respectively. More strikingly, the combination of aspirin and γT reduced the overall tumor area by 60% (P < 0.05). Noteworthy, the supplementation of γT also alleviated aspirin-induced stomach lesion and appeared to modulate intestinal microbial composition. Our study demonstrated that the combination of aspirin and γT has stronger anticancer activity than aspirin or γT alone while alleviates aspirin-associated adverse effect, suggesting that the combination of γT and aspirin is a more effective and safer chemopreventive agent for CRC than aspirin alone.</p>

La barrière intestinale est la plus grande surface de contact entre le milieu extérieur et le milieu intérieur. Outre ses fonctions d'absorption des nutriments, elle exerce un rôle important de défense contre les agents indésirables (toxines, bactéries) contenus dans la lumière intestinale. Une augmentation de la perméabilité intestinale a été observée chez les patients atteints du syndrome de l'intestin irritable (SII) ou des maladies inflammatoires chroniques de l'intestin (MICI). Cette hyperperméabilité intestinale est contemporaine d'une hypersensibilité viscérale à la distension de la paroi intestinale. Des travaux récents rapportent également une augmentation de l'activité protéolytique du contenu intestinal dans le cadre de ces deux pathologies. Les estrogènes, par leurs propriétés anti-inflammatoires et leur capacité à moduler la perméabilité intestinale par activation de leurs récepteurs (REs) peuvent contribuer à l'amélioration des symptômes associés à ces pathologies digestives. Une variété de traitements médicaux a été utilisée pour la prise en charge thérapeutique du SII et de MICI. Cependant, les patients questionnent les cliniciens sur des conseils diététiques susceptibles d'améliorer leur qualité de vie. Ainsi, l'objectif de ce travail était d'évaluer les effets et les mécanismes d'action impliqués, d'un traitement par du germe de soja fermenté (SG) sur l'hyperalgésie viscérale et l'hyperperméabilité intestinale dans des modèles animaux mimant le SII et les MICI afin de proposer des futures allégations santé à ce produit. Le rationnel de l'évaluation de cet ingrédient était basé sur sa composition intéressante, à savoir, sa teneur en composés à propriétés estrogéniques (isoflavones) et sa capacité à inhiber les protéases (BBI). Dans un premier temps, nous avons montré qu'un traitement oral de 15 jours par le SG diminue de façon significative l'hypersensibilité viscérale, l'hyperperméabilité intestinale ainsi que l'augmentation de l'activité protéolytique induites par un stress de contrainte chez le rat. La diminution de la perméabilité intestinale implique une surexpression de l'occludine, protéine des jonctions serrées. De même, le traitement par du SG réduit la densité des mastocytes au niveau du côlon. Tous les effets préventifs du SG sauf ceux sur l'activité protéolytique sont estrogéno-dépendants car bloqués par l'antagoniste des REs. Dans un second temps, nous avons montré qu'un traitement préventif par le SG pendant 15 jours présente des effets protecteurs vis-à-vis d'une inflammation intestinale induite par du TNBS. Le SG atténue la sévérité de l'inflammation, l'hyperperméabilité, l'hypersensibilité et l'augmentation de l'activité protéolytique induites par la colite. Les effets anti-inflammatoires du SG sont à la fois dépendants des phytoestrogènes et du contenu de l'ingrédient en BBI. En conclusion, ces données sont prometteuses pour une future utilisation du SG dans la gestion thérapeutique du SII et des MICI comme traitement adjuvant / The intestinal barrier is the largest area of contact between the external environment and internal environment. In addition to its function of nutrient absorption, the intestinal barrier plays a key role of defense against noxious agents (toxins, bacteria) contained in the intestinal lumen. An increase in intestinal permeability was observed in patients with irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD). This intestinal hyperpermeability was often associated with visceral hypersensitivity to colorectal distension. Recent studies also report an increase in the proteolytic activity in patients with IBS or IBD. Estrogens, through their anti-inflammatory properties and their ability to modulate intestinal permeability by activating estrogen receptors (ERs), can play an important role in these digestive diseases. A variety of medical therapies have been used for treatment of IBS and IBD. However, patients question clinicians about dietary suggestions to improve their symptoms and quality of life. Thus, the aim of this study was to evaluate the effects and mechanisms of action involved of a treatment with fermented soy germ (SG) on visceral hyperalgesia, intestinal hyperpermeability in animal models mimicking the IBS and IBD. The evaluation of this ingredient was based on its interesting composition, i.e its content of isoflavones and a family of serine protease inhibitors known as BBI. Initially, we demonstrated that an oral treatment of 15 days by SG significantly reduces visceral hypersensitivity, intestinal hyperpermeability and increased proteolytic activity induced by acute stress in the rat. Decreased intestinal permeability is due to overexpression of occludin, a transmembrane tight junction protein. Similarly, treatment with SG reduces the density of colonic mast cells. All preventive effects of SG except those on the proteolytic activity are estrogen-dependent because blocked by the antagonist of ERs. In a second step, we demonstrated that a treatment for 15 days with SG induces protective effects against intestinal inflammation induced by TNBS. SG reduces the severity of colitis, decreases TNBS-induced hyperpermeability, hypersensitivity and increased proteolytic activity. The anti-inflammatory effects of SG are estrogen and/or BBI dependent. In conclusion, these data are promising for future use of the SG as adjuvant therapy in IBS and IBD management

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<p>The gut and brain are involved in a bi-directional communication system, referred to as the gut-brain axis. While it has been established that antimicrobials induce dysbiosis in the gut, which further disrupts immune and metabolic homeostasis, research on brain and behaviour development is becoming a topic of interest. We propose that alterations via antibiotics at the level of the gut microbiota impacts the gut-brain axis. The primary interest of this thesis is to understand the effects that antibiotics have on brain and behaviour development in conjunction with changes in the immune system and metabolism using the antibiotic mouse model. Mice treated with antibiotics revealed behavioural differences in the open field apparatus and three-chamber social behaviour apparatus, but not in the elevated plus maze and auditory fear conditionings enclosures. Evaluation of intestinal permeability revealed that female Balb/C mice administered a combination of bacitracin, neomycin and primaricin and another group administered a combination of ampicillin, neomycin and primaricin showed reduced intestinal permeability. Furthermore, the immune system condition was evaluated using flow cytometric analysis of spleens, which revealed no effect of treatment on immune cell profiles in CD1 mice treated with ampicillin. Evaluation of serum cytokine levels showed minimal differences in Balb/C and C57Bl/6 mice treated with antibiotics. Body weight and water and food consumption were evaluated in mice administered antibiotics. Weight loss differences were observed in two groups of female Balb/C mice, with the first group administered bacitracin, neomycin and primaricin and the second group administered ampicillin , neomycin and primaricin. Antibiotic treatment dependent differences in water and food consumption were observed. Serum insulin and leptin level investigation revealed that female Balb/C mice administered ampicillin, neomycin and primaricin had reduced serum insulin levels compared to strain matched controls. These findings indicate that antibiotic treatment impact metabolic function. This pilot study using antibiotic treated mouse models provides insight on the microbiota’s effects on the gut-brain axis, which can help to potentially identify methods of preventing gut microbiota mediated pathology in humans.</p> / Master of Science (MSc)

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zdeněk Záboj Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of drugs interactions of antiviral drugs with intestinal transporters Sofosbuvir is an antiviral agent widely used in the treatment of chronic hepatitis C. This orally administered prodrug is a designed substrate of ATP-binding (ABC) efflux transporters, P- glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2). ABCB1 and ABCG2 are important determinants of intestinal absorption and are the site of significant pharmacokinetic drug interactions, leading to changes in drug exposure. Pharmacokinetic drug interactions may be undesirable (increasing the toxicity of the treatment) or desirable (allowing dose reduction). Because sofosbuvir is often administered in combination regimens with other anti(retro)virotics, the aim of this thesis was to study the ability to enhance intestinal absorption of sofosbuvir. To study the pharmacokinetic drug interactions on ABCB1 and ABCG2, a widely established in vitro bi-directional transport method through a polarized monolayer formed by the Caco-2 cell line derived from colorectal cancer has been used. We analyzed the drug interactions of sofosbuvir on these efflux...