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Identifying Mechanisms Used by Adherent-invasive Escherichia coli Associated with Crohn Disease to Evade the Immune SystemOssa, Juan C. 15 August 2012 (has links)
Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation.
Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used.
Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting.
Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades.
Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.
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Identifying Mechanisms Used by Adherent-invasive Escherichia coli Associated with Crohn Disease to Evade the Immune SystemOssa, Juan C. 15 August 2012 (has links)
Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation.
Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used.
Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting.
Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades.
Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.
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E. coli adhérentes et invasives et pathogénèse de la maladie de Crohn : rôle du facteur hypoxique HIF-1 / Non disponibleMimouna, Sanda 29 October 2013 (has links)
La maladie de Crohn (MC) est une maladie inflammatoire chronique intestinale (MICI). Son incidence et sa prévalence ont augmenté en Europe au cours des dix dernières années (150 pour 100000 habitants) constituant ainsi un problème de santé majeur. L’inflammation chronique dans la MC favorise la mise en place d’une angiogenèse pathophysiologique. Inflammation et angiogenèse sont deux réponses cellulaires suspectées dans la survenue des cancers coliques associés au MICI. Même si les facteurs favorisant la mise en place de la MC restent non élucidés, la contribution des bactéries exogènes est fortement suspectée. Parmi ces bactéries, les E.coli adhérentes et invasives (AIEC), isolées à partir de la muqueuse iléale de patients porteurs de la MC, sont un bon candidat. Les objectifs de mon projet de thèse étaient de caractériser les mécanismes moléculaires induits par les AIEC et impliqués dans la mise en place des réponses pro inflammatoire et pro angiogénique des cellules intestinales épithéliales. Le facteur de transcription hypoxique (HIF-1) est au cœur de l’immunité innée et de l’angiogenèse. J’ai émis l’hypothèse que les AIEC pouvaient moduler le niveau d’expression de HIF-1α et ainsi contrôler les réponses pro inflammatoire et pro angiogénique. Dans mon premier article, j’ai montré que HIF-1α est maximalement exprimé au niveau de l’épithélium iléal des patients porteurs de la MC. Ensuite, j’ai montré sur un modèle murin compétent pour l’infection par les AIEC, les souris CEABAC10, que les bactéries induisent l’augmentation du niveau protéique de HIF-1 α ainsi que l’activation de la voie de signalisation du VEGF, le facteur angiogénique le plus puissant. / Non communiqué.
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Sonoporation de cellules adhérentes par cavitation inertielle régulée / Adherent cells sonoporation by regulated inertial cavitationLabelle, Pauline 20 November 2014 (has links)
La sonoporation, c'est-à-dire l'utilisation d'ultrasons pour augmenter la perméabilité de la membrane cellulaire et permettre le transfert de molécules dans la cellule, est une méthode de transfection alternative intéressante. Cependant, même s'il est généralement admis que la cavitation acoustique joue un rôle important dans la sonoporation, les mécanismes physiques sous-jacents à ce phénomène ne sont pas totalement compris. Pour obtenir des informations sur les interactions entre les bulles, les cellules et le milieu environnant, nous avons développé un système de sonoporation adapté à la visualisation en temps-réel sous microscope et dédié aux cellules adhérentes. Le champ acoustique dans le puits cellulaire est composé d'ondes stationnaires et possède donc des positions d'équilibres pour les bulles au fond du puits, c'est-à-dire à proximité des cellules. Après avoir confirmé que les effets biologiques sont liés à la cavitation inertielle, un système de régulation de la cavitation inertielle a été implémenté pour augmenter la reproductibilité de la sonoporation. L'utilisation de cette régulation permet de s'affranchir des problèmes d'initiation et de maintien de l'activité de cavitation ainsi que de travailler sans ajout d'agents de contraste ultrasonore. Ce système permet de sonoporer des cellules adhérentes et ceci de manière plus reproductible lors de l'utilisation de la régulation qu'à intensité acoustique fixée. L'utilisation de la régulation permet également de s'affranchir de la température du milieu (à 24 ou 37 °C). De plus, la sonoporation des cellules ne semblent pas induire d'effets négatifs sur la reprise de croissance des cellules. L'utilisation de membrane modèle (des bicouches lipidiques fluorescentes) permet l'observation des interactions bulles-membrane, principalement sous la forme de dégâts de type fissures ou impacts présents à la fois sur les ventres et nœuds de pression acoustique. Ces positions particulières sont également le siège du détachement cellulaire et de la sonoporation / Sonoporation, the use of ultrasound to increase cell membrane permeability and allow the transfer of molecules into cells, is an interesting alternative method of transfection. However, even if it is generally admitted that acoustic cavitation plays an important role in sonoporation, the physical mechanisms acting during sonication are not fully understood. To obtain information on the interaction between bubbles, cells and the _owing medium during sonication, we designed a sonoporation device adapted to real-time microscope visualization and dedicated to adherent cells. The acoustic field in the well is composed by standing waves and provides cavitation bubble equilibrium positions at the bottom of the well, near cells. After biological effects have been confirmed to be linked to inertial cavitation, a regulation device on inertial cavitation has been implemented in order to improve reproducibility of sonoporation. This regulation allows overcoming problems linked to initiation and time stability of cavitation activity without adding ultrasound contrast agents in the medium. The sonoporation device allows the sonoporation of adherent cells, and this, with more reproducible results when using regulation instead of a fixed acoustic intensity. The cavitation control allows also to obtain the same biological effects at 24 and 37 °C. Furthermore, cell sonoporation does not apparently induce negative effects on cell growth. The use of a membrane model (fluorescent lipid bilayer) allows the observation of bubbles-membrane interactions, principally in the form of damages as cracks or impacts present at both nodal and anti-nodal positions of the acoustic field. Cell detachment and sonoporation appear also at these particular locations
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Evaluation of Intestinal Microbial Diversity and a New Antibiotic Regimen in Crohn's Disease PatientsAlcedo, Karel 01 January 2015 (has links)
Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs – Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.
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Targeting Cancer Stem-LIike Cells in Human Esophageal Squamous Carcinoma Cell Lines by CurcuminAlmanaa, Taghreed N. 16 December 2013 (has links)
No description available.
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The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coliSnelling, Anna M., Macfarlane-Smith, Louissa, Fletcher, Jonathan N., Okeke, Iruka N. 2009 December 1921 (has links)
Yes / Background. The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed.
Results. We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have
contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies.
Conclusions. A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.
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Caracterização do perfil de compostos orgânicos voláteis produzidos por cultura de células e animais infectados com Leishmania infantum / Characterization of the profile of volatile organic compounds produced by cell cultures and animals infected with Leishmania infantumAnchieta, Naira Ferreira 19 December 2016 (has links)
Alguns parasitas modificam o perfil de Compostos Orgânicos Voláteis (COVs) de seus hospedeiros na tentativa de atrais/dispersar seus vetores, assegurando a propagação do parasita e a manutenção do ciclo da doença. É sabido que o mosquito Lutzomyia longipalpis, vetor da Leishmaniose Visceral (LV) são mais atraídos por cães infectados que não infectados. Este trabalho tem como objetivo identificar e caracterizar os Compostos Orgânicos Voláteis emitidos por culturas de células e hamsters infectados Mesocricetus auratus com Leishmania infantum (MHOM/74/PP75). Células aderentes mononucleares de baço de hamsters foram separadas com Ficoll e distribuídas em placas de 24 poços com 2x106 células por poço. Após 42 horas, as células foram infectadas e os COVs foram coletados por \"headspace\" por 18 horas. Os COVs foram determinados por comparação com as placas controle: controle células aderentes mononucleares, controle L. infantum, e controle meio RPMI 1640.40 Hamsters foram subdivididos em 2 grupos: 20 animais foram infectados com formas promastigotas metacíclicas de L. infantum selecionadas com aglutinina de amendoim (PNA) por via intracardíaca com 1x107 parasitas. Os animais foram acompanhados por 4 meses pós infecção e as amostras foram coletadas colocando os animais em sacos de poliéster conectados com um tubo de metal recheado com Tenax TA acoplado a uma bomba de sucção automática por 10 minutos. Todas as amostras foram analisadas por dessorção térmica via Cromatografia gasosa/ Espectrometria de Massas (CG-EM). Nas culturas de células, 2 compostos estão presentes apenas nas culturas infectadas: 1,2-difenilciclobutano e (E)-(2,3-Difenil Ciclopropil)-metil-fenilsulfóxido. O 2,4-dimetilbenzaldeído estava presente na placa controle contendo meio RPMI 1640. Por outro lado, em ambas as placas infectadas e controle com células aderentes mononucleares, o 3-metileno-heptano estava presente. Outros compostos contendo os grupos químicos aldeídos ou álcoois ou hidrocarbonetos aromáticos foram detectados em todas as placas, mas com diferentes áreas de pico (quantidade). Quando comparamos os COVs emitidos por hamsters infectados e controles, nenhuma diferença foi encontrada. Os compostos emitidos por culturas de células infectadas mostram que a infecção com L.infantum é capaz de modificar o perfil de COVs em culturas de células. Outros compostos encontrados em todas as placas, mas com diferentes áreas de pico podem indicar que a infecção aumenta a liberação de algumas substâncias que são possíveis atrativos para vetores. / Some parasites can modify their hosts Volatile Organic Compounds (VOCs) profile in order to attract / disperse their vectors, ensuring spreading of the parasite and maintenance of the disease cycle. It is already known that Lutzomyia longipalpis sandflies, the vectors of Visceral Leishmaniasis (VL), are more attracted to infected than to non-infected dogs. This work aims to identify and characterize the VOCs emitted by cell cultures and hamsters Mesocricetus auratus infected with Leishmania infantum (MHOM/74/PP75). Spleen adherent mononuclear cells of hamsters were separed with Ficoll and distributed in flat-bottomed 24-well plates at 2x106 cells per well. After 42 hours, the cells were infected and VOCs were collected from the \"headspace\" for 18 hours. The VOCs were determined in comparison with the controls plates: control mononuclear adherent cells, control L. infantum and control medium RPMI 1640. 40 Hamsters were subdivided in two groups: 20 animals were used as a control group and 20 animals were infected with metaciclic form of L. infantum selected by peanut agglutinin (PNA) by intracardiac route with 1x107 parasites. The animals were followed up for 4 months post-infection and the samples were collected by introducing the hamsters in a polyester bag connected with a metal tube filled with the Tenax TA adsorbent coupled to an automatic suction pump for 10 minutes. All samples were analyzed by thermal desorption via Gas Chromatography/Mass Spectrometry (GCMS). In cell cultures, two compounds were present only in infected cultures: 1, 2- diphenyl cyclobutane, and trans-[(2, 3-Diphenylcyclopropyl) methyl] phenyl sulfoxide. 2, 4-dimethyl benzaldehyde was present in the control well containing RPMI 1640 medium. On the other hand, in both infected and control wells with mononuclear adherent cells, 3-methylene heptane was present. Other compounds containing either aldehydes or alcohols or aromatic hydrocarbon chemical groups were detected in all plates, but with different peak areas (i.e., amounts). When comparing the VOCs emitted by infected and control hamsters, no difference was found. The compounds emitted only by infected cell cultures show that the infection with L. infantum is able to modify the profile of VOCs in cell cultures. The other compounds found in all plates but with different peak areas may indicate that infection may increase the release of some substances that are possible attractants for vectors.
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Stratégies thérapeutiques visant à limiter les E.coli Adhérents-Invasifs du tractus digestif dans le cadre de la Maladie de Crohn. / Prophylactic and therapeutic strategies to limit Adherent-Invasive Escherichia coli (AIEC) in the digestive tract in the context of Chron's diseaseSivignon, Adeline 30 June 2015 (has links)
La maladie de Crohn (MC) est une maladie inflammatoire chronique du tube digestif caractérisée par un état d’hyperactivation du système immunitaire intestinal. Les données cliniques et expérimentales montrent que l’étiologie de la MC serait une réponse immunitaire aberrante à des facteurs environnementaux et/ou infectieux chez un hôte génétiquement prédisposé. Les patients atteints de MC présentent une perméabilité intestinale anormalement élevée pouvant expliquer la stimulation du système immunitaire intestinal par les bactéries et antigènes du microbiote. La muqueuse iléale des patients atteints de MC est anormalement colonisée par des souches de Escherichia coli ayant la propriété d’adhérer et d’envahir les cellules épithéliales intestinales, de survivre et de se multiplier dans les macrophages en entraînant la sécrétion de TNF-α (Tumor Necrosis Factor-alpha). Un pathovar de E. coli associé à la MC et dénommé AIEC pour « Adherent-Invasive E. coli » a été défini. Les souches AIEC adhérent via l’adhésine FimH des pili de type 1 aux résidus mannose de la glycoprotéine CEACAM6, anormalement exprimée au niveau de l’épithélium iléal des patients atteints de MC. Au cours de la maladie, l’inflammation intestinale peut être contrôlée par les traitements médicamenteux ou la chirurgie sans pour autant obtenir de rémission complète et définitive.Le but du travail était d’analyser les conséquences de l’infection par des bactéries AIEC in vivo dans un modèle murin reproduisant l’interaction AIEC/CEACAM6 et de proposer des stratégies pour éliminer ces bactéries de l’intestin. Nous avons montré que les AIEC avaient la capacité d’altérer la fonction de barrière de l’épithélium intestinal chez des souris transgéniques CEABAC10 exprimant CEACAM6. Cette altération est associée à une forte induction de l’expression de la protéine de jonction Claudine-2, comme observée chez les patients atteints de MC. Nous avons ensuite démontré que la levure S. cerevisiae CNCM I-3856 et des produits de levures étaient capables de maintenir l’intégrité de la barrière intestinale des souris en prévenant la colonisation du tractus digestif par les bactéries AIEC. Dans une deuxième partie, nous nous sommes intéressés au développement de nouvelles molécules antagonistes de l’adhésine FimH. Les thiazolylaminomannosides ont montré un puissant effet inhibiteur de l’adhésion des bactéries AIEC à des cellules épithéliales intestinales. Les heptyl-mannosides sont également de puissants inhibiteurs de l’adhésion des AIEC et leur présentation en multivalence sur des corps polymériques ou des cyclodextrines potentialisent l’effet anti-adhésif. De manière intéressante, certains heptyl-mannosides diminuent fortement la colonisation de l’intestin par les bactéries AIEC et préviennent la colite chez des souris CEABAC10. Toutefois, la multivalence n’apporte pas d’efficacité supplémentaire dans ce contexte. En conclusion, deux stratégies anti-adhésives ont été étudiées : les levures et les mannosides. Elles pourraient être proposées aux patients atteints de MC fortement colonisés par les AIEC afin d’éliminer ces bactéries du tractus digestif pour espérer diminuer l’inflammation. Les perspectives à ce travail seront la mise au point de techniques de détection simples et rapides des personnes colonisées ou susceptibles d’être colonisées par ces souches de E. coli pour cibler la population à traiter par les probiotiques levures et les molécules anti-adhésives. / Crohn’s disease (CD) is an inflammatory bowel disease (IBD) with a multifactorial etiology, resulting from an exacerbated inflammatory response to intestinal microbes and/or microbial components in genetically susceptible hosts. CD patients present an increased intestinal permeability which can favor the overstimulation of the intestinal immune system by bacteria or antigens from microbiota. Ileal mucosa from CD patients is abnormally colonized by Escherichia coli strains sharing the ability to adhere to and to invade intestinal epithelial cells, to survive and to replicate within macrophages, inducing high secretion of TNF-α (Tumor Necrosis Factor-). These strains associated with CD are grouped in a pathovar of E. coli named AIEC for « Adherent-Invasive E. coli ». AIEC bacteria adhere via the adhesin FimH localized at the tip of the type 1 pili, to mannose residues exposed on the glycoprotein CEACAM6 abnormally expressed at the ileal mucosa of CD patients. Currently, intestinal inflammation can be controlled with drugs or intestinal surgery but total remission cannot be yet achieved. The aim of the present work was to investigate consequences of AIEC infection in a murine model mimicking the AIEC/CEACAM6 interaction and to test different strategies to eradicate these bacteria from the gut. We showed that AIEC bacteria altered barrier function of the intestinal epithelium in transgenic CEABAC10 mice expressing human CEACAM6. The overexpression of the pore-forming tight junction protein claudin-2 was correlated with the increase intestinal permeability, as observed in CD patients. We demonstrated that the yeast strain S. cerevisiae CNCM I-3856, as well as some yeast products, were able to prevent increase of intestinal permeability in decreasing AIEC gut colonization. In a second part, we investigated another strategy targeting AIEC bacteria using antagonists to FimH adhesin. Thiazolylaminomannosides molecules exerted a strong inhibitory effect on the ability of AIEC bacteria to adhere to intestinal epithelial cells. Heptyl-mannosides (HM) also shared high inhibitory properties in vitro and their efficacy can be potentiated when HM are harbored in multiple copies on polymeric or cyclodextrin cores. Interestingly, some HM molecules strongly decreased AIEC gut colonization and the signs of colitis in vivo, in AIEC LF82-infected CEABAC10 mice. In that context, multivalency did not improve inhibitors efficacy.To conclude, two different strategies were studied: probiotic yeasts and anti-adhesive mannosides. These treatments should be proposed in CD patients highly colonized by AIEC bacteria in order to eliminate these bacteria from the gut and to decrease intestinal inflammation. Future works will focus on the development of quick and easy detection methods to determine people colonized or susceptible to be colonized by AIEC bacteria to treat this subpopulation of CD patients.
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Fluorinated pickering emulsions for droplet-based microfluidics technology / Emulsions fluorées de Pickering pour la technologie de microfluidique en gouttesChacon Orellana, Laura A. 23 July 2018 (has links)
Les émulsions fluorées de Pickering sont étudiées et mises au point dans la technologie demicrofluidique en gouttes pour des applications d’études sur des cellules adhérentes isolées.Les principaux résultats de ce projet sont : l’établissement d’un lien entre la couverture desurface des nanoparticules et la fluidité de l’émulsion de Pickering ; l’établissement deslignes directrices pour la stabilisation des gouttes avec un débit de production élevé et unminimum de déchets de particules ; et la mise en oeuvre d’une plateforme technologiquecomplète pour l’étude des cellules RPE, pour mesurer leur hétérogénéité phénotypique auniveau de la cellule individuelle. / Fluorinated Pickering emulsions are studied and engineered within droplet-based microfluidicstechnology for adherent-cell studies applications. The main findings of this projectinclude: linking the nanoparticles surface coverage to the bulk flowability of the Pickeringemulsion; deriving guidelines for droplet stabilization with high production throughput andminimal particle waste; and implementing the full technological platform for the study ofRPE cells, while unraveling their phenotypic heterogeneity at the single cell level.
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