In the intestinal epithelium, Wnt signaling drives the expression of the genes encoding tyrosine kinase receptors EphB2 and EphB3 and represses the expression of their membrane-tethered ligands, ephrin-Bs. Eph-ephrin interactions result in cellular repulsion and are involved in boundary formation. The project of this thesis is to understand the mechanism by which EphB−ephrin-B signals restrict cell positioning of cell types (cell sorting) in the normal intestinal epithelium and suppress colorectal cancer (CRC) progression beyond the earliest stages. We have demonstrated that at the onset of CRC EphB receptors impair the expansion of tumor cells through a mechanism dependent on E-cadherin–mediated adhesion. We show that EphB-mediated compartmentalization restricts the spreading of EphB+ tumor cells into ephrin-B1+ territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1+ intestinal cells at the onset of tumorigenesis. We have discovered that cell sorting is the outcome of two integrated mechanisms: cell contraction/repulsion and differential cell adhesion. The latter is the driving force to induce EphB/ephrin-B−mediated cell compartmentalization. We have developed in vitro models to analyze the mechanisms that induce E-cadherin remodeling upon EphB activation. We found RhoA, p120-catenin and the metalloproteinase ADAM10 as downstream effectors of EphB signaling involved in the control of cell sorting in CRC cells. / A l'epiteli intestinal, la ruta de senyalització Wnt indueix l'expressió dels gens que codifiquen per als receptors tirosina kinasa EphB2 i EphB3 i reprimeixen la dels seus lligands transmembrana, efrines de tipus B. Les interaccions Eph-efrina causen repulsió cel·lular i estan implicades en la formació de fronteres entre compartiments. La finalitat d'aquesta tesi és entendre el mecanisme pel qual la senyalització per EphB−efrina-B restringeix el posicionament dels diferents tipus cel·lulars a l'epiteli intestinal normal i suprimeix la progressió del càncer colorectal (CRC) en els primer estadis. Hem demostrat que, a l’inici del CRC, els receptors EphB restringeixen l'expansió de les cèl·lules tumorals a través d'un mecanisme depenent d'adhesió intercel·lular a través d’E-cadherina. En aquest treball es mostra in vitro i in vivo que la compartimentalització mitjançada per la senyalització dels receptors EphB restringeix l’invasió de les cèl·lules tumorals EphB+ als territoris efrina-B+. Aquests resultats indiquen que les cèl·lules de CRC han de silenciar l’expressió d'EphB per evitar les interaccions repulsives imposades per les cèl·lules intestinals normals efrina-B+ circumdants al començament del procés de tumorigènesi. Hem pogut discernir que el reordenament cel·lular per senyals EphB−efrina-B és el resultat de dos mecanismes integrats: la contracció/repulsió intercel·lular i l’adhesió diferencial entre diferents poblacions cel·lulars. Aquesta última és la força principal que condueix a la compartimentalització cel·lular mitjançada per EphB−efrina-B. Hem desenvolupat models in vitro per analitzar els mecanismes que provoquen el remodelament de la E-cadherina sota la senyalització per EphB. Presentem RhoA, p120-catenina i ADAM10 com a efectors de la senyalització de la ruta EphB implicats en el control de la compartimentalització cel·lular en el CRC.

Microbiota colonisation of the intestinal tract makes it difficult for pattern recognition receptors (PRR) to discriminate between beneficial microbes and harmful pathogens. We aim to define the roles of cytosolic Nod-like receptors (NLR) in intestinal immunity and homeostasis. Upon activation, some NLR form inflammasomes that mediate the release of inflammatory cytokines and pyroptosis, an inflammatory form of cell death. NLR activation in the non-hematopoietic compartment was shown to be protective during acute intestinal infection. To identify the cell type responsible for this protection, we generated transgenic mice in which the key inflammasome adaptor molecule Asc is selectively ablated in intestinal epithelial cells (IEC) (Asc^ΔVC) and observed that inflammasomes are important for controlling Citrobacter rodentium clearance in these mice. To further dissect the importance of pathogen clearance by IEC inflammasome, ex vivo cultures of primary IEC organoids were established. Thus far this system has revealed profound differences in inflammasome regulation between IEC organoids and bone marrow-derived macrophages (BMDM). This research will inform our understanding of cell type-specific regulation of inflammasomes.

Les maladies inflammatoires de l’intestin (MIIs, [MIM 266600]) sont caractérisées par une inflammation chronique au niveau du tube gastro-intestinal. Les deux principales formes sont la maladie de Crohn (MC) et la colite ulcéreuse (CU). Les MIIs résulteraient d’un défaut du système immunitaire et de l’épithélium intestinal. Ce dernier forme une barrière physique et biochimique qui sépare notre système immunitaire des microorganismes commensaux et pathogènes de la microflore intestinale. Un défaut dans la barrière épithéliale intestinale pourrait donc mener à une réponse immunitaire soutenue contre notre microflore intestinale. Les études d’association pangénomiques (GWAS) ont permis d’identifier 201 régions de susceptibilité aux MIIs. Parmi celles-ci, la région 1q32 associée à la MC (p<2x10-11) et à la CU (p<6x10-7) contient 4 gènes, dont C1orf106, un gène codant pour une protéine de fonction inconnue. Le re-séquençage de la région 1q32 a permis d’identifier une variante génétique rare de C1orf106 (MAF˂1%) associée aux MIIs (p=0,009), Y333F. Nous avons démontré que la substitution de la tyr333 par une phénylalanine semble avoir un effet sur la stabilité protéique de C1orf106 tel que démontré lors de l’inhibition de la synthèse protéique induite par le cycloheximide. Nous avons déterminé que C1orf106 est exprimé dans le côlon et l’intestin grêle. De plus, son expression est augmentée lors de la différenciation des cellules épithéliales Caco-2 en épithélium intestinal polarisé. Son profil d’expression correspond aux types cellulaires et tissulaires affectés dans les MIIs. De plus, C1orf106 est partiellement co-localisée avec le marqueur des jonctions serrées, ZO-1. Toutefois, son marquage reproduit parfaitement celui du marqueur des jonctions adhérentes, E-cadhérine. Les jonctions serrées et adhérentes sont localisées du côté apical de la jonction intercellulaire et sont toutes deux impliquées dans l’établissement de la barrière épithéliale. Nous avons donc testé l’impact de C1orf106 sur la perméabilité de l’épithélium intestinal. Nous avons observé une augmentation de la perméabilité épithéliale chez un épithélium intestinal formé par des cellules Caco-2 sous-exprimant C1orf106. Nos résultats suggèrent que C1orf106 pourrait être le gène causal de la région 1q32. / The Inflammatory bowel diseases (IBD, [MIM 266600]) involve chronic inflammation of the digestive tract and include ulcerative colitis (UC) and Crohn’s disease (CD). IBD may result from defects in the homeostasis of immune system and intestinal epithelium. The latter forms a physical and biochemical barrier to commensal and pathogenic microorganisms. A dysfunction in the epithelial barrier may lead to a sustained immune response against the gut flora. Genome wide association studies (GWAS) have identified 200 susceptibility regions in IBD. Among these, the 1q32 region associated with risk of both CD (p<2x10-11) and UC (p<6x10-7), contains the gene C1orf106. Our targeted re-sequencing study has identified a low-frequency variant, Y333F (p=0.009) in C1orf106, a protein of unknown function and in which tyrosine333 is predicted to be phosphorylated. We demonstrated that its substitution by a phenylalanine may have an effect on C1orf106 protein stability as shown by cycloheximide treatment experiments. Our RNA expression analyses of human tissues and cell lines demonstrated that C1orf106 is mostly expressed in the small intestine and colon. It is also detectable in monocytic cell lines but more highly expressed in colonic epithelial cell lines. Furthermore, its expression is increased by 40% during differentiation of colonic epithelial Caco-2 cells into polarized epithelium. To provide further biological context, we generated colorectal LS174T cells that stably overexpress the Y333F alleles and demonstrated that it is partially localized with ZO-1, used as a tight junction (TJ) marker. We did observe tighter colocalization with E-cadherin, a canonical marker for adherens junctions (AJ), typically located below the TJ complex. AJ and TJ play an essential role in the establishment of epithelial barrier. The localization of C1orf106 at these regions suggests its possible implication in epithelial barrier homeostasis. Using trans-epithelial measurement of ions movement across epithelium, we demonstrated an increased in permeability of an epithelium formed by C1orf106 knock-down Caco-2 cells. Our results suggest that C1orf106 could be the causal gene of the 1q32 susceptibility region.

Métodos in vitro utilizando epitélio intestinal animal são importantes ferramentas para avaliar a permeabilidade de fármacos, propriedade que é um importante parâmetro de biodiosponibilidade. Considerando que o maior objetivo na indústria farmacêutica é desenvolver novos fármacos com boa biodisponibilidade oral, o projeto teve como objetivo padronizar o modelo de permeação com membrana de intestino de rã (Rana catesbeiana) em células de Franz, comparando seus resultados com ensaios de células Caco-2. Os fármacos modelo utilizados foram os antivirais zidovudina e aciclovir. A quantidade de fármaco permeado foi determinada por método de eletroforese capilar para o método com intestino de rã touro e por HPLC-UV para os ensaios com células Caco-2. O parâmetro de permeação foi o coeficiente de permeabilidade aparente (Papp) dos fármacos para ambos modelos experimentais. Para estabelecimento do protocolo experimental dos estudos de permeabilidade intestinal de rã, foi proposto um projeto fracionado 24-1 com 4 ensaios adicionais usando o software Minitab, e as variáveis foram: secção intestinal, pH da solução de Ringer e temperatura. A análise do planejamento experimental feita pela estimativa dos parâmetros da regressão obtidos através dos resultados do modelo fatorial possibilitou a determinação dos coeficientes da equação matemática que definiu a influência das variáveis sobre o coeficiente de permeabilidade aparente dos fármacos. Os efeitos das variáveis pH e temperatura interpretados conjuntamente apresentaram interferência leve, porém as variáveis fármaco e secção intestinal interpretados juntos tiveram interferência importante, mostrando maior permeação dos fármacos através da secção inicial do intestino da rã. Os resultados de Papp foram: para o metoprolol, pelo método das células Caco-2 foi de 28 x 10-6 cm/s, valor que está de acordo com demais dados de células Caco-2 na literatura e o valor obtido com células de Franz que mais se adequada a estes resultados e demais disponíveis provenientes de outras técnicas na literatura, foi de 28,1 x 10-6 cm/s, em uma das condições do planejamento estatístico utilizando segmento final da membrana epitelial da rã. No caso do aciclovir, o resultado de Papp de 0,48 x 10-6 cm/s também obtido em uma das condições envolvendo segmento intestinal final da rã foi exatamente igual a o valor 0,48 x 10 - 6 cm/s, encontrado com células Caco-2 no presente estudo e estão de acordo com outros valores disponíveis na literatura por Trapani e colaboradores, 2004 e também com células Caco-2. Para zidovudina o valor de Papp obtido em uma das condições utilizando segmento intestinal inicial da rã, de 13 x 10-6 cm/s, foi a que mais se assemelhou ao obtido pela técnica de células Caco-2, 13,6 x 10-6 cm/s, e também está de acordo com demais dados da literatura. / There are lots of different in vitro technics in the literature using animal intestinal epithelium to estimate permeability of drugs, property that is an important parameter of bioavailabity. Considering that the main objective of Pharmaceutical Companies is the development of new drugs with good oral bioavailabity, the aim of this work was to standardize the permeability of antivirals using in vitro/ex vivo method of intestinal epithelium of Rana catesbeiana in Franz cells and compare these results to those obtained from studies using Caco-2 cells. Zidovudine and Acyclovir were selected as model drugs. The amount of drug permeated will be determined by the method of capillary electrophoresis for assays using Rana Catesbeiana and HPLC-UV for studies with Caco-2 cells. The permeation parameter determined was the apparent permeability coefficient (Papp) of drugs for both experimental models. To establish the experimental protocol to studies of intestinal permeability of frog, it was proposed a fractional 24-1 design with 4 additional tests using Minitab software and the variables were: intestinal section, pH of Ringer solution and temperature. The analysis of the experimental design made by the estimate of the regression parameters obtained from the factorial model results allowed the determination of the coefficients of the mathematical equation that defines the influence of the variables on the apparent permeability coefficient of acyclovir and zidovudine. The effects of pH and temperature interpreted jointly presented a slight interference, but the variables drug and intes tinal section interpreted together had major interference, showing greater permeation of drugs through the initial section of the intestine of the frog . The results of Papp were: for metoprolol, with the method of Caco-2 cells was 28 x 10-6 cm/s, a value which is consistent with other data of Caco-2 cells provided in the literature and the condition obtained with Franz cells that are most suitable for these and other results obtained from other techniques available in the literature, was 28.1 x 10-6 cm/s, provided with the final intestinal segment using frog epithelial membrane. In the case of acyclovir, the result of Papp of 0.48 x 10-6 cm/s obtained in one condition with final frog intestinal segment was exactly equal to the value of 0.48 x 10-6 cm/s, found with Caco-2 cells in the present study and are in agreement with other values available in the literature for Trapani and colegues, 2004 and also with Caco-2 cells. The Papp value for zidovudine obtained with the initial gut segment of frog, 13 x 10-6 cm /s was which more resembled that obtained by the technique of Caco-2 cells, 13.6 x 10-6 cm/s and is also consistent with other literature data.

Métodos in vitro utilizando epitélio intestinal animal são importantes ferramentas para avaliar a permeabilidade de fármacos, propriedade que é um importante parâmetro de biodiosponibilidade. Considerando que o maior objetivo na indústria farmacêutica é desenvolver novos fármacos com boa biodisponibilidade oral, o projeto teve como objetivo padronizar o modelo de permeação com membrana de intestino de rã (Rana catesbeiana) em células de Franz, comparando seus resultados com ensaios de células Caco-2. Os fármacos modelo utilizados foram os antivirais zidovudina e aciclovir. A quantidade de fármaco permeado foi determinada por método de eletroforese capilar para o método com intestino de rã touro e por HPLC-UV para os ensaios com células Caco-2. O parâmetro de permeação foi o coeficiente de permeabilidade aparente (Papp) dos fármacos para ambos modelos experimentais. Para estabelecimento do protocolo experimental dos estudos de permeabilidade intestinal de rã, foi proposto um projeto fracionado 24-1 com 4 ensaios adicionais usando o software Minitab, e as variáveis foram: secção intestinal, pH da solução de Ringer e temperatura. A análise do planejamento experimental feita pela estimativa dos parâmetros da regressão obtidos através dos resultados do modelo fatorial possibilitou a determinação dos coeficientes da equação matemática que definiu a influência das variáveis sobre o coeficiente de permeabilidade aparente dos fármacos. Os efeitos das variáveis pH e temperatura interpretados conjuntamente apresentaram interferência leve, porém as variáveis fármaco e secção intestinal interpretados juntos tiveram interferência importante, mostrando maior permeação dos fármacos através da secção inicial do intestino da rã. Os resultados de Papp foram: para o metoprolol, pelo método das células Caco-2 foi de 28 x 10-6 cm/s, valor que está de acordo com demais dados de células Caco-2 na literatura e o valor obtido com células de Franz que mais se adequada a estes resultados e demais disponíveis provenientes de outras técnicas na literatura, foi de 28,1 x 10-6 cm/s, em uma das condições do planejamento estatístico utilizando segmento final da membrana epitelial da rã. No caso do aciclovir, o resultado de Papp de 0,48 x 10-6 cm/s também obtido em uma das condições envolvendo segmento intestinal final da rã foi exatamente igual a o valor 0,48 x 10 - 6 cm/s, encontrado com células Caco-2 no presente estudo e estão de acordo com outros valores disponíveis na literatura por Trapani e colaboradores, 2004 e também com células Caco-2. Para zidovudina o valor de Papp obtido em uma das condições utilizando segmento intestinal inicial da rã, de 13 x 10-6 cm/s, foi a que mais se assemelhou ao obtido pela técnica de células Caco-2, 13,6 x 10-6 cm/s, e também está de acordo com demais dados da literatura. / There are lots of different in vitro technics in the literature using animal intestinal epithelium to estimate permeability of drugs, property that is an important parameter of bioavailabity. Considering that the main objective of Pharmaceutical Companies is the development of new drugs with good oral bioavailabity, the aim of this work was to standardize the permeability of antivirals using in vitro/ex vivo method of intestinal epithelium of Rana catesbeiana in Franz cells and compare these results to those obtained from studies using Caco-2 cells. Zidovudine and Acyclovir were selected as model drugs. The amount of drug permeated will be determined by the method of capillary electrophoresis for assays using Rana Catesbeiana and HPLC-UV for studies with Caco-2 cells. The permeation parameter determined was the apparent permeability coefficient (Papp) of drugs for both experimental models. To establish the experimental protocol to studies of intestinal permeability of frog, it was proposed a fractional 24-1 design with 4 additional tests using Minitab software and the variables were: intestinal section, pH of Ringer solution and temperature. The analysis of the experimental design made by the estimate of the regression parameters obtained from the factorial model results allowed the determination of the coefficients of the mathematical equation that defines the influence of the variables on the apparent permeability coefficient of acyclovir and zidovudine. The effects of pH and temperature interpreted jointly presented a slight interference, but the variables drug and intes tinal section interpreted together had major interference, showing greater permeation of drugs through the initial section of the intestine of the frog . The results of Papp were: for metoprolol, with the method of Caco-2 cells was 28 x 10-6 cm/s, a value which is consistent with other data of Caco-2 cells provided in the literature and the condition obtained with Franz cells that are most suitable for these and other results obtained from other techniques available in the literature, was 28.1 x 10-6 cm/s, provided with the final intestinal segment using frog epithelial membrane. In the case of acyclovir, the result of Papp of 0.48 x 10-6 cm/s obtained in one condition with final frog intestinal segment was exactly equal to the value of 0.48 x 10-6 cm/s, found with Caco-2 cells in the present study and are in agreement with other values available in the literature for Trapani and colegues, 2004 and also with Caco-2 cells. The Papp value for zidovudine obtained with the initial gut segment of frog, 13 x 10-6 cm /s was which more resembled that obtained by the technique of Caco-2 cells, 13.6 x 10-6 cm/s and is also consistent with other literature data.

Colorectal cancer is one of the most common cancer types worldwide. Both genetic and epigenetic alterations play a critical role in its initiation and progression. One of the genes frequently epigenetically silenced or lost in many types of human cancer is tumor suppressor gene Hypermethylated in Cancer 1 (HIC1). It encodes for transcriptional repressor regulating its target genes directly or indirectly. Twelve genes whose expression is repressed by HIC1 have been identified to date. These genes encode for transcription factors, cell cycle and apoptosis regulators or proteins involved in angiogenesis as well as cell migration and invasiveness. Employing mouse embryonic fibroblasts upon Hic1-conditional knockout we have revealed six novel genes potentially repressed by Hic1 including Toll-like receptor 2 (Tlr2). Here we show that Tlr2 is one of the Hic1 target genes and that Hic1 inactivation in the intestine leads to increased Tlr2 production. Moreover, enhanced inflammatory response upon chemical-induced colitis as well as increased tumor formation in ApcMin mice was observed in Hic1-deficient mice. Expression profiling in human fibroblast upon HIC1 knockdown revealed increased expression of another potential target gene, transcription factor E2F7. Our study describes a new relationship between HIC1 and...

Colon cancer causes approximately seven percent of all cancer-related deaths in the world and presumably due to modern lifestyle, it is also one of the most frequently diagnosed cancers. The inefficiency of standard treatment indicates the need for intensive research of molecular mechanisms of cancer development. The canonical Wnt signaling pathway is essential for maintenance of the progenitor phenotype of stem cells in crypts of the intestine and controls repopulation of the epithelia, in physiological conditions. However, aberrant activation leads to tumor formation. Although Wnt signaling in cancer has been subjected to thorough investigation, there is still a lot of questions concerning further branching of the pathway. As a model of Wnt/β-catenin triggered colorectal cancer, we use mice with mutated APC, which is the tumor suppressor involved in this pathway. Previous expression profiling of the intestinal tumors from relevant mice revealed two transcription factors: ETV4 and MSX1 which are significantly overexpressed in cancer cells. In this project we elucidate whether the overexpression is really tumor restricted and Wnt dependent or there is a crosstalk with another signal transduction pathway. We investigate the function and regulation of these transcription factors by synthetic reporter assays,...

The canonical Wnt signaling pathway is one of the most important pathways involved in cell proliferation and differentiation. It is highly conserved in evolution and participates not only in embryonic development but also in adult tissue homeostasis. In the intestine, Wnt signaling is closely connected to maintenance of intestinal stem cells and renewal of the epithelia. Conversely, aberrant activation of the Wnt signaling pathway underlies different types of human diseases. Its constitutive activation results in neoplasia and specifically in development of colorectal cancer, which is the third most common malignancy in western world. The aim of this thesis was to uncover various aspects of the regulatory mechanisms of the Wnt/β-catenin signaling cascade. Furthermore, I headed to find novel Wnt pathway modulators and confirm their function in vivo. The results are presented in four publications. The first study examines murine Wnt proteins processing and the sequential order of Wnt post-translational modifications which are required for the secretion and signaling activity of the ligands. Next publication focuses on the gene Troy, which we identified as negative regulator of Wnt signaling. TROY was discovered as a Wnt target gene during DNA microarray profiling of human colorectal cancer cells....

L’administration par voie orale des médicaments reste encore de nos jours la voie royale de la prise des médicaments car moins onéreuse et plus adaptée au confort du patient. Mais cette voie reste toujours inaccessible pour certains médicaments comme les médicaments biologiques et les bio similaires voir certains anticancéreux et antirétroviraux.Le but de ce travail est d’améliorer la biodisponibilité par voie orale des médicaments à faible biodisponibilité par la mise au point d’un promoteur d’absorption. Pour y arriver nous avons adopté comme stratégie de développer un promoteur qui agit à la fois sur le passage passif et sur le passage actif des médicaments. Les études in vitro ont été réalisées en chambre de perméation d’Ussing adaptées par la société Biomécatronics SAS (BéthuneFrance). Dans la première partie de ce travail (Brevet), nous avons montré que l’utilisation d’une composition pharmaceutique et/ou diététique comprenant un extrait de plante(Hibiscus sabdariffa) pouvait augmenter la biodisponibilité in vitro des médicaments et des xénobiotiques qui passent par la voie paracellulaire comme le cisplatine (21 fois),l’oxaliplatine (11fois), la fluorescéine isothiocyanate-Dextran 4000 (3 fois), mais également les médicaments connus pour leur transport actif par la voie transcellulaire comme l’Efavirenz (7 fois) et l’Atazanavir (4 fois). Dans la seconde partie de ce travail, nous avons cherché à vérifier si notre promoteur d’absorption des médicaments a un effet sur la couche de mucus intestinale.Cette couche peut être un facteur limitant de passage des médicaments au travers de la barrière intestinale.Dans un premier temps (article 1), nous avons induit l’augmentation de la couche mucus au niveau du colon de rat après un prétraitement pendant une semaine avec le métronidazole. Puis nous avions confirmé (article 2) que l’administration par voie orale de deux antibiotiques le Cotrimoxazole (CTX) et le métronidazole (MTZ) pendant une semaine augmente la couche de mucus au niveau du côlon ; aussi nous avons montré qu’il existe une relation entre l’augmentation de la couche de mucus et la diminution de la conductance qui est l’index de transport passif des ions, des électrolytes et de certaines molécules à faibles poids moléculaires.De plus l’augmentation de la couche de mucus au niveau de l’intestin est responsable de la diminution du passage transépithélial des deux antirétroviraux dont l’utilisation est recommandée en première ligne par l’OMS (le.Ritonavir et l’Atazanavir) surles sujets porteurs du VIH (virus de l’immunodéficience humain). Après les traitements auMTZ et au CTX la sécrétion de l’Atazanavir augmente respectivement dans le côlon proximal de 2 et 4 fois et dans le côlon distal de 3 et 5 fois. On obtient également une sécrétion du Ritonavir de 5 et 10 fois dans le proximal et de 2 et 5 fois plus dans le distal.Le travail se poursuit par l’étude de l’effet de notre promoteur d’absorption des médicaments sur la couche de mucus intestinal.En conclusion, ce travail montre que l’on peut augmenter la biodisponibilité in vitroen utilisant les promoteurs de l’absorption des xénobiotiques qui agissent à la fois au niveau du transport passif et actif. / Oral dosing is still seen as the silver bullet of drug administration, as it is cheaper andbetter adapted to patient comfort. However, oral route is still inaccessible to many drugssuch as biologics and biosimilars respectively certain anticancer drugs and antiretrovirals(ARV).The aim of this present study was to find new drugs enhancers that improve the oralbioavailability of drugs and xenobiotics. All the studies were realized in vitro using Ussingchambers technic. To achieve the set objective we used the strategy to develop drugenhancer which can modulate at the same time transcellular and paracellular pathways.In the first part of this study (patent) we have shown that the use of a pharmaceutical and /or a dietetic formulation containing a plant extract (Hibiscus sabdariffa) could increase thebioavailability in vitro in rats not only of cisplatin (21 fold), oxaliplatin (11 fold) andFluorescein Isothiocyanate-Dextran 4000 (FD4, 3 fold). All that drugs were transportedthrough intestinal barrier using paracellular pathway. In addition the study showed thatthis formulated enhancer can increased the bioavailability of Efavirenz (7 fold) andAtazanavir (4 fold) which are active transported.In order to assess the effect of new drugs enhancer on mucus thickness that limits thetransport of xenobiotic through intestinal barrier, we decide to evaluate his effect on passiveand active transport of drugs.In the second part of this study we have shown that after a week of pre-treatment of ratswith Metronidazole (MTZ, publication 1) and Cotrimoxazole (CTX, publication 2), the twomost commonly used antibiotics in the prophylaxis against opportunistic infections in HIV /AIDS, both increase colonic mucus thickness that affect directly passive intestinalpermeability by reducing conductance an index of passive transport through intestinalepithelium. In addition those antibiotics also entail a change in the transepithelialconductance and ARV fluxes. After MTZ and CTX treatment the secretion of Atazanavir(ATZ) increases respectively in the proximal colon by 2 to 4 fold and in the distal colon by 3to 5 fold respectively. Ritonavir (RTV) is poorly absorbed in control, after a week of pretreatmentwith MTZ and CTX one rather notices a secretion of RTV 5 to 10 fold higher in theproximal and 2 to 5 fold higher in the distal colon. The next study will be conducted toevaluate the effect of new drugs enhancer on mucus thickness layer.In conclusion, oral bioavailability of drugs and xenobiotics can be enhanced bypharmaceutical composition that contains herbal extract which increase passive and activetransport of drugs through intestinal barrier.

Background &amp; Aims: Celiac disease (CD) is a chronic inflammatory small-bowel enteropathy caused by permanent intolerance to gliadin in wheat gluten, and related proteins in ray and barley. It is disputed whether CD patients tolerate oats. The only treatment of CD is lifelong gluten-free diet (GFD). Only individuals that carry the HLA-DQ2 and/or DQ8 alleles, and eat gluten can develop CD. Dysbiosis in the gut microbiota is a suggested risk factor for CD. T cells in small intestinal mucosa, including intraepithelial lymphocytes (IELs), are known to be important in the pathogenesis of CD. In contrast, the role of intestinal epithelial cells (IECs) is poorly understood. In this thesis we investigated the role of IECs in the immune pathology of CD from duodenal mucosa of children with CD, clinical controls and treated CD. We also investigated the role of CD associated bacteria and oats supplemented GFD on the mucosal immune system. Results: A new CD-associated bacterium, Prevotella jejuni, was isolated and characterized. It is a saccharolytic and proteolytic anaerobe. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were upregulated in IECs in active CD. In two in vitro models for intestinal epithelium, small intestine enteroids and T84 polarized tight monolayers, we showed that 70% of these genes were upregulated by interferon (IFN)-γ via the IRF1 pathway. IRF1 was also upregulated by the CD-associated bacteria P. jejuni and Actinomyces gravenitzii. IECs expressed the NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin (IL)-18, which induces IFN-γ in IELs. P. jejuni bound the intestinal epithelial cell lines T84, Caco2, HT29, and INT407, while Lachnoanaerobaculum umeaense preferentially bound Caco2. P. jejuni caused decreased transepithelial resistance over tight monolayers, while L. umeaense caused an increase. P. jejuni upregulated mRNAs for the detoxification molecules CYP1A1, CYP1A2, CYP1B1, and TIPARP, the chemokines CX3CL1, CXCL1, and CXCL10, the sialyltranserase ST3GAL4, and the inflammation promoting protein S100A3 in tight monolayers. L. umeaense upregulated the chemokines CCL20 and CXCL10, and down-regulated TLR2. In a randomized, double-blinded intervention trial comparing two study-groups, standard GFD and oat-containing GFD, we found that mRNAs for several immune effector molecules and tight junction proteins were only reduced in patients receiving GFD, but not in a substantial fraction of patients on GFD with oats. The down-regulatory cytokines IL-10 and TGF-β1, the cytotoxicity-activating NK-receptors NKG2C and NKG2E, and the tight junction protein claudin-4 remained elevated in the study group on GFD with oats. Conclusions: IECs are far from inactive in CD. A key factor in the epithelial reaction in CD appears to be over-expression of IRF1 in IECs. Dual activation of IRF1 and IRF1-regulated genes, both directly by P. jejuni and indirectly by IFN-γ via the IL-18-inflammasome, would drastically enhance the inflammatory response and lead to the pathological situation seen in active CD. P. jejuni harms the intestinal epithelium, i.e., it is a likely risk factor for CD, while L. umeaense strengthen barrier function and local immunity, possibly acting as a protective. A fraction of CD patients should avoid oats in the diet. / Doctoral thesis