Tous les spectres RMN utilisées comme exemples sont tirés d&#39;une étude publiés antérieurement 7 qui a été soutenu financièrement par Nestlé.

1. La colonisation du germe sans animaux et la collecte de l&#39;échantillon <ol><li> Retirer les animaux sans germes d&#39;isolateurs et de les héberger dans une salle de l&#39;élevage dans des cages conventionnelles équipés d&#39;un filtre à l&#39;avant des animaux conventionnels qui servent de témoins (figure 1). </li><li> Mélanger la moitié de la litière (3 jours) prises de la cage de commande classique avec la litière des animaux sans germes. Toujours garder un tiers de la litière sale conventionnels chaque fois qu&#39;il est nécessaire de le renouveler afin de maintenir un niveau de bactéries (il conserve pendant au moins 3 jours). </li><li> Recueillir l&#39;urine dans un microtube de 1,5 ml par une manipulation de la souris sur le tube et aider à la miction par masser doucement l&#39;intestin. Snap-geler immédiatement dans l&#39;azote liquide. Stockez au moins à -40 ° C jusqu&#39;à l&#39;analyse par RMN. Un volume minimum de 20 uL est nécessaire pour l&#39;acquisition d&#39;une sonde RMN de 5 mm, mais il est recommandé d&#39;utiliser 30 uL d&#39;améliorer la qualité de profilage métabolique. </li><li> Les animaux doivent be euthanasié sans l&#39;utilisation d&#39;anesthésiques afin d&#39;éviter les résonances RMN confusion due à un métabolisme hépatique de composés anesthésiques (par exemple, utiliser dislocation cervicale suivie d&#39;une confirmation de la mort par exsanguination) </li></ol> 2. Recommandation pour la collecte de la biopsie du foie <ol><li> Ne pas utiliser tout produit contenant de l&#39;alcool pour éviter toute contamination. Laver les outils utilisant la solution d&#39;eau ou une solution saline. </li><li> Ne pas percer la vésicule biliaire. En cas de fuite biliaire, laver immédiatement avec de l&#39;eau des tissus ou de solution saline. </li><li> Recueillir des biopsies du foie (environ 15-50 mg) dans le lobe gauche, comme illustré dans la figure 2. Pour les biopsies reproductibles, de collecter de manière cohérente dans le centre du lobe gauche en évitant les zones périphériques où le tissu est plus mince. </li><li> Aligner les biopsies geler dans l&#39;azote liquide immédiatement et les stocker à -80 ° C jusqu&#39;à l&#39;analyse par RMN. </li></ol> 3. RMN 1H acquisition de l&#39;urine microvolume </p&gt; <ol><li> Préparer 0,2 M solution de tampon phosphate de sodium dans D2O (99,8%), pH 7,4 contenant 1 mM 3 - (triméthylsilyl) d&#39;acide propionique 4-D (TSP). </li><li> Mélanger 30 uL d&#39;urine avec 30 uL de tampon phosphate de sodium. </li><li> Transférer 50 ul de solution mixte dans 1,7 mm RMN tube capillaire (figure 3 (2)) à l&#39;aide d&#39;une seringue en verre de 50 uL équipé d&#39;une aiguille métallique (OD 0,5 mm). Soyez prudent pour éviter les bulles. </li><li> Monter l&#39;adaptateur capillaire (Figure 3 (3)) sur le dessus de l&#39;échantillon d&#39;urine contenant des capillaires et le placer dans un microtube de 2,5 mm pour la RMN de 5 mm Sonde RMN (figure 3) (1). Utilisez cette combinaison de tubes en tant que régulier 5 mm pour tube RMN acquisition spectrale. </li><li> Utilisez la tige d&#39;extraction (Figure 3 (4)) pour enlever les capillaires de 2,5 mm tube RMN en vissant l&#39;adaptateur capillaires doucement pour la retirer. </li></ol> . 4 1 H RMN HR MAS de tissu biopsie du foie: la préparation des échantillons <ol><li> MAS composants du rotor et les outils sontdécrit dans la figure 4. </li><li> Insérer une biopsie (environ 15-50 mg) dans le rotor de zirconium (figure 4) (1) et remplissez le reste du volume avec pure O 2 D pour la RMN serrure. Attention à ne pas faire remonter les bulles car cela altérer la qualité du calage ultérieur et la qualité de l&#39;acquisition de données. </li><li> Insérer entretoise 50 uL Téflon (figure 4 (2)) en utilisant les vis cylindrique (figure 4 (5)). Dévissez et c&#39;est calibrer en utilisant la jauge de profondeur sur le côté court (figure 4 (8)). À cette étape, il est important de prêter une attention particulière à l&#39;échantillon, car une partie de cela peut fuir à travers le trou d&#39;espacement. Si cela est le cas, alors une partie de la biopsie est détruit et le poids de l&#39;échantillon n&#39;est plus fiable. Il est donc nécessaire de recommencer depuis le début de la préparation de l&#39;échantillon. </li><li> Placez la broche thead (figure 4 (3)) et vissez doucement avec le tournevis (figure 4 (6)). Sécher l&#39;eau résiduelle avec un morceau de tissu. </li><li> Placez le couvercle (Figure 4 (4)) à lasupérieur du rotor et l&#39;insérer dans l&#39;emballeur du rotor (figure 4 (6)). Appuyez fermement jusqu&#39;à ce que le bouchon est en place. Il ne doit pas être tout espace laissé entre le rotor et son capuchon. </li><li> Mark moitié de la partie inférieure du rotor en utilisant un marqueur noir pour permettre la détection optique de vitesse de rotation. </li><li> Placer le rotor à l&#39;intérieur du spectromètre RMN et commencer à tourner à 5 kHz. Acquérir spectre RMN 1H utilisant CPMG séquence d&#39;impulsions 13, conformément aux directives du fabricant. </li><li> Utilisez la résonance α glucose anomérique à 5,22 ppm (doublet) pour calibrer les spectres RMN. </li><li> Pour déballer le rotor, procéder en enlevant le bouchon en utilisant le déménageur bouchon (figure 4 (9)). Dévissez et retirez thead broches téflon entretoise en utilisant la vis cylindrique. Laver abondamment à l&#39;eau et de détergent. </li></ol> 5. Les résultats représentatifs L&#39;activité microbienne intestinale peut être contrôlée en utilisant le profilage métabolique urinaire. Un grand nombre de microbes urinairesco-métabolites identifiables par RMN 1H ont été décrits dans la littérature 7,14-17. Ces microbes de co-métabolites sont particulièrement utiles pour surveiller le processus de colonisation car ils fournissent un moyen rapide et non invasive pour estimer quand l&#39;écosystème nouvellement établie est stable. La figure 5A illustre clairement l&#39;apparition d&#39;microbienne intestinale co-métabolites sur le processus de colonisation. Cette figure montre un profil métabolique urinaire obtenu en suivant la procédure décrite à l&#39;étape 2 pour un animal colonisé 20 jours en utilisant la procédure décrite à l&#39;étape 1. Cet animal n&#39;a pas excréter le sulfate de toute indoxyle et les montants très peu de phenylacetylglycine (PAG) et p-crésol sulfate à l&#39;état de germe de frais (jour 0-bleu). Comme la colonisation progresse, ces trois marqueurs du métabolisme des protéines par le microbiote intestinal augmente considérablement pour atteindre un équilibre à jour 20 (rouge). Ceci est particulièrement facile à suivre pour un groupe d&#39;animaux, comme illustré sur la figure 5B en utilisant le PAGrésonance. Ce diagramme a été obtenue en intégrant la surface sous les résonances surlignées en gris sur la figure 5A (δ 7,40 à 7,43), ce qui correspond à une résonance particulière (triplet) de GCP pour un groupe de 7 animaux. 1 H à haute résolution Magic Angle Spinning (MAS RH) La spectroscopie RMN est une technique non destructive qui permet des acquisitions rapides et reproductibles de profils métaboliques de tout type de biopsie de 18 ans. Dans ce protocole, nous avons utilisé cette technique puissante pour obtenir un profil métabolique hépatique de deux souris avant (bleu) et après (rouge) colonisation (figure 6). Ce chiffre illustre bien l&#39;information qui peut être dérivé d&#39;un profil métabolique RMN MAS-fondée. De nombreux acides aminés ainsi que des métabolites issus de métabolisme énergétique comme le glucose, le glycogène, le lactate, les triglycérides, (D)-3-hydroxybutyrate et nicotinurate peuvent être visualisées. Ces profils contiennent également des informations pertinentes au stress oxydatif (c&#39;est à dire un ascorbiquecid, le glutathion), métabolisme des nucléotides (c&#39;est à dire l&#39;inosine, uridine) et le métabolisme de la méthylamine (c.-choline, la triméthylamine-N-oxyde). Dans cet exemple, il est très clair que le germe sans souris affiche presque pas de glycogène et de très faibles quantités de glucose et de triglycérides comme cela a été précédemment publiées 7. <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="Figure 1."/> Figure 1. Présentation du protocole colonisation. Animaux axéniques et conventionnelles sont logés dans des cages équipées de filtres côté à côte et leurs portées sont échangées afin de permettre la colonisation progressive du microbiote intestinal conventionnel (1). L&#39;activité microbienne intestinale est surveillé en utilisant 1 H RMN profilage métabolique basé (2-3). Le métabolisme hépatique est évalué par une MAS RH H profilage métabolique par RMN basé (4-5). <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="Figure 2."/> Figure 2. Souris vivanteAnatomie r. Le foie est affiché comme du côté plat de l&#39;orgue fait face à la table. Pour les biopsies reproductibles, il est conseillé de toujours prélever des échantillons à partir du centre du lobe gauche, comme indiqué par le rectangle en pointillés. <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="Figure 3."/> Figure 3 1,7 mm RMN kit capillaire pour travailler avec microvolumes clés:.. 1: 2,5 microtube RMN mm, 2: 1,7 mm RMN tube capillaire, 3: adaptateur capillaire, 4: tige d&#39;extraction. <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="Figure 4."/> Équipements MAS Figure 4 rotors principaux:.. 1: rotor MAS, 2: 50 uL entretoise en téflon, 3: thead broches, 4: CAP, 5: Vis cylindrique, 6: tournevis, 7: rotor conditionneur, 8: jauge de profondeur. <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="Figure 5."/> Figure 5. Évolution des profils métaboliques urinaires durant colonization. <ol class="ualpha"><li> Zoom sur la région aromatique des spectres entre 6.8 à 7.8 ppm, où la co-métabolites microbiens peuvent être visualisées. Spectres RMN 1 H ont été obtenues à partir d&#39;un seul individu au jour 0 (bleu), 4 (vert), 15 (orange) et 20 (rouge) l&#39;après-colonisation. La zone grise correspond à la zone qui a été intégrée pour rendre le schéma B. clés: 1-MeHistamine: 1-méthylhistamine; indoxyl-S: le sulfate de Indoxyl;:; Son p-crésol-S Histidine: le sulfate de p-crésol; PAG : Phenylacetylglycine. </li><li> La concentration moyenne durant la colonisation PAG (n = 7). T de Student-test a été utilisé pour comparer la différence de concentration en PAG à divers points de temps: a: p &lt;0,05 par rapport au jour 0, b: p &lt;0,01 par rapport à 10 jours. </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="Figure 6."/> Figure 6. Typique de 600 MHz 1 H HR spectres RMN MAS de biopsies du foie provenant germ-free (en bleu) et ex-germ-free (red) les souris. Protons en gras sont responsables de la résonance de triglycérides clés:. 3-HB: 3-hydroxybutyrate, le GSH: glutathion réduit, TG: triglycérides, OTMA: triméthylamine-N-oxyde.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+partial+least+squares+models+and+related+chemometric+approaches+in+metabonomics+and+metabolic+phenotyping.">The evolution of partial least squares models and related chemometric approaches in metabonomics and metabolic phenotyping.</a> Journal of Chemometrics. 24, 636-649 (2010).</li><li>Merrifield, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+system-wide+characterisation+of+the+pig:+a+model+for+human+physiology.">A metabolic system-wide characterisation of the pig: a model for human physiology.</a> Mol. Biosyst. , (2011).</li><li>Tugnoli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+human+gastric+mucosa+by+HR-MAS+magnetic+resonance+spectroscopy.">Molecular characterization of human gastric mucosa by HR-MAS magnetic resonance spectroscopy.</a> International Journal of Molecular Medicine. 14, 1065-1071 (2004).</li><li>Sitter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HR+MAS+MR+spectroscopic+profiles+of+breast+cancer+tissue+with+clinical+parameters.">Comparison of HR MAS MR spectroscopic profiles of breast cancer tissue with clinical parameters.</a> NMR Biomed. 19, 30-40 (2006).</li><li>Beckonert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling,+metabolomic+and+metabonomic+procedures+for+NMR+spectroscopy+of+urine,+plasma,+serum+and+tissue+extracts.">Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.</a> Nat. Protoc. 2, 2692-2703 (2007).</li></ol>

Nous n&#39;avons rien à révéler.

Dans ce protocole, nous avons décrit une procédure de colonisation progressive dans un milieu ouvert aux autres d&#39;étudier l&#39;impact du microbiote intestinal sur le métabolisme hépatique évalué par 1 H RMN HR MAS profilage des intacte biopsie. Diverses méthodes de la colonisation ont été décrits dans la littérature. Les méthodes les plus communes de coloniser les animaux avec un microbiote sont définis par gavage oral ou de l&#39;eau potable contaminée 19,20. L&#39;inoculation ...

Table des réactifs spécifiques et des équipements: <table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> 2,5 mm microtube </td><td> Nouvelle ère </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 mm tube capillaire </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Adaptateur capillaire </td><td> Nouvelle ère </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> Extraction tige </td><td> Nouvelle ère </td><td> NE-341-5 </td><td></td></tr><tr><td> HR-MAS BL4 rotor avec 50 pl sphériques kit entretoise en téflon </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> Kit d&#39;outils pour 50 inserts uL </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> Advance III 600RMN MHz </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H RMN du solide HR MAS Sonde </td><td> Bruker </td><td></td><td></td></tr><tr><td> L&#39;oxyde de deutérium 99,9% </td><td> Sigma-Aldrich </td><td> 530867-1L </td><td></td></tr><tr><td> 3 - (triméthylsilyl) propionique acide D-4 (TSP) </td><td> Sigma-Aldrich </td><td> 269913 </td><td></td></tr></tbody></table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

Il est bien connu que les bactéries intestinales contribuent de manière significative à l&#39;homéostasie hôte, offrant une gamme de prestations telles que la protection immunitaire et la synthèse de vitamine. Ils fournissent également l&#39;hôte d&#39;une quantité considérable de nutriments, ce qui rend cet écosystème un organe métabolique essentiel. Dans le contexte de plus en plus évident le lien entre la flore intestinale et le syndrome métabolique, la compréhension des interactions métaboliques entre l&#39;hôte et sa microflore intestinale est devenue un enjeu important de la biologie moderne. 1-4 Colonisation (appelé aussi processus de normalisation) désigne la création de micro-organismes dans une ancienne exempte de germes d&#39;origine animale. S&#39;il est un processus naturel survenant à la naissance, il est aussi utilisé chez les adultes sans germes animaux pour contrôler l&#39;écosystème intestinal floraux et de mieux déterminer son impact sur le métabolisme de l&#39;hôte. Une procédure commune pour contrôler le processus de colonisation est d&#39;utiliser la méthode de gavage avec un single ou un mélange de micro-organismes. Cette méthode conduit à une colonisation très rapide et présente l&#39;inconvénient d&#39;être extrêmement stressant 5. Il est donc utile de minimiser le stress et d&#39;obtenir un processus plus lent de colonisation d&#39;observer progressivement l&#39;impact de la mise en place sur le métabolisme des bactéries hôtes. Dans ce manuscrit, nous décrivons une procédure pour évaluer la modification du métabolisme hépatique au cours d&#39;un processus de colonisation progressive en utilisant une technique non destructive métaboliques profilage. Nous proposons de suivre la colonisation microbienne intestinale en évaluant l&#39;activité métabolique microbienne intestinale reflétée par l&#39;excrétion urinaire de métabolites microbiens co-par une profilage métabolique par RMN H-fondée. Cela permet une appréciation de la stabilité de l&#39;activité microbienne intestinale delà de l&#39;établissement stable de l&#39;écosystème microbien intestinal généralement évaluée par le suivi des bactéries fécales par DGGE (électrophorèse sur gel dénaturant gradient). 6 Lela colonisation se déroule dans un environnement ouvert et conventionnelles est initiée par une litière sale souillés par des animaux conventionnels, qui serviront de témoins. Les rongeurs sont des animaux coprophages, ce qui assure une colonisation homogène, comme décrit précédemment. 7 Profilage métabolique hépatique est mesurée directement à partir d&#39;une biopsie du foie intact en utilisant 1 H High Angle magique Résolution Spinning spectroscopie RMN. Cette technique semi-quantitative offre un moyen rapide d&#39;évaluer, sans endommager la structure cellulaire, les principaux métabolites tels que les triglycérides, de glucose et de glycogène afin de mieux estimer l&#39;interaction complexe entre les processus de colonisation et le métabolisme hépatique 7-10. Cette méthode peut également être appliqué à toute une biopsie tissulaire 11,12.

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Une procédure est décrite colonisation progressive pour mieux évaluer son impact sur le métabolisme hépatique hôte. La colonisation est surveillé non invasive, en évaluant l&#39;excrétion urinaire de métabolites microbiens co-par RMN basée sur le profilage métabolique tandis métabolisme hépatique est évalué par la Haute Résolution angle magique (MAS RH) RMN de profilage intacte biopsie.

Évaluer les changements métaboliques hépatiques pendant la colonisation progressive des axéniques de souris par 1 H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

assessing-hepatic-metabolic-changes-during-progressive-colonization

Research

JoVE Journal

Immunology and Infection

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

13.0K vues.  The University of Reading. Une procédure est décrite colonisation progressive pour mieux évaluer son impact sur le métabolisme hépatique hôte. La colonisation est surveillé non invasive, en évaluant l'excrétion urinaire de métabolites microbiens co-par RMN basée sur le profilage métabolique tandis métabolisme hépatique est évalué par la Haute Résolution angle magique (MAS RH) RMN de profilage intacte biopsie.

Vidéo: Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

Quantification de la colonisation fongique, sporogénèse, et la production de mycotoxines utilisant Bioassays noyau

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Recherche

Immunologie et infection

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

La colonisation de Euprymna scolopes Squid par Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Détection des microARN dans la microglie par PCR en temps réel dans le SNC normal et pendant neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caractérisation des réponses inflammatoires au cours de la colonisation intranasale avec Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Évaluation de l&#39;activité anti-fongique isolé macrophages alvéolaires par microscopie confocale

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Évaluation de l’invasion bactérienne des cellules cardiaques en culture et colonisation cardiaque chez des souris infectées à l’aide de Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Récolte sélective de Marginating-hépatiques Leucocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modèle murin du Groupe B<em&gt; Streptococcus</em&gt; Vaginal Colonisation

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Diffusion moléculaire Membranes plasmatiques de Lymphocytes primaires mesurées par Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Détection de la colonisation de Escherichia Coli entéro-hémorragique en hôte murin par système de Bioluminescence Non invasif In Vivo

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...