Alle NMR-Spektren als anschauliche Beispiele verwendet werden, aus einer zuvor veröffentlichten Studie 7, die finanziell von Nestlé unterstützt wurde abgeleitet.

1. Colonization von keimfreien Tieren und Probenentnahme <ol><li> Entfernen keimfreien Tieren aus Isolatoren und Haus sie in einem konventionellen Tierhaltung Raum in Käfigen mit Filter vor der konventionellen Tiere, die als Kontrollen (Abbildung 1) dienen ausgestattet. </li><li> Die Hälfte des Wurfes (3 Tage alt) von der Steuerung konventioneller Käfig mit dem Wurf des keimfreien Tieren. Halten Sie immer 1 / 3 der schmutzigen herkömmlichen Wurf jedes Mal ist es notwendig, sie zu erneuern, um auf ein Niveau von Bakterien (halten Sie sie mindestens 3 Tage) zu erhalten. </li><li> Sammeln Sie Urin in ein 1,5 ml Mikroröhrchen durch den Umgang mit der Maus über das Rohr und helfen Miktion durch sanfte Massage des Darmes. Snap-freeze sofort in flüssigem Stickstoff. Shop zumindest bei -40 ° C bis NMR-Analyse. Ein Mindestvolumen von 20 ul ist für den Erwerb mit einem 5 mm NMR-Sonde erforderlich, aber es wird empfohlen, 30 ul zu verwenden, um die Qualität der Metabolic Profiling zu verbessern. </li><li> Die Tiere sollten be ohne Verwendung von Betäubung getötet, um Verwechselung-NMR-Signale durch den Leberstoffwechsel der Narkose Verbindungen (z. B. Verwendung Genickbruch durch eine Bestätigung des Todes durch Ausbluten folgt) zu vermeiden </li></ol> 2. Empfehlung für die Sammlung der Leberbiopsie <ol><li> Verwenden Sie keine Produkte, die Alkohol, um eine Kontamination zu vermeiden. Wash-Tools nur mit Wasser oder Kochsalzlösung. </li><li> Nicht durchlöchern Gallenblase. Im Falle der Galle Leck, waschen Gewebe sofort mit Wasser oder Kochsalzlösung. </li><li> Sammeln Leberbiopsien (etwa 15-50 mg) aus dem linken Lappen, wie in Abbildung 2 dargestellt. Für reproduzierbare Biopsien, konsequent zu sammeln in der Mitte des linken Lappens Vermeidung der peripheren Gebieten, in denen Gewebe wird dünner. </li><li> Snap freeze Biopsien in flüssigem Stickstoff sofort und lagern Sie sie bei -80 ° C bis NMR-Analyse. </li></ol> 3. 1 H NMR Erwerb von Urin Mikrovolumens </p&gt; <ol><li> Bereiten 0,2 M Natrium-Phosphat-Puffer-Lösung in D2O (99,8%), pH 7,4 mit 1 mM 3 - (Trimethylsilyl)-propionsäure-d 4 (TSP). </li><li> Mix 30 ul Urin mit 30 ul Natriumphosphatpuffer. </li><li> Transfer-50 ul gemischte Lösung in 1,7 mm-NMR-Kapillare (Abbildung 3) (2) mit einem 50 ul Glasspritze mit einer Metall-Nadel (OD 0,5 mm) ausgestattet. Seien Sie vorsichtig, um Blasen zu vermeiden. </li><li> Fit der Kapillare Adapter (Abbildung 3) (3) auf der Oberseite der Kapillare mit Urinprobe und legen Sie sie in einen 2,5-mm-NMR-Mikroröhrchen für 5 mm NMR-Sonde (Abbildung 3 (1)). Mit dieser Kombination aus Röhren als regulärer 5 mm NMR-Röhrchen für die spektrale Akquisition. </li><li> Verwenden Sie die Extraktion Stange (Abbildung 3) (4) zur Kapillare von 2,5 mm NMR-Röhrchen zu entfernen durch Eindrehen der Kapillare Adapter vorsichtig, um ihn herauszuziehen. </li></ol> . 4 1 H HR-MAS-NMR von Lebergewebe Biopsie: Probenvorbereitung <ol><li> MAS-Rotor-Komponenten und Werkzeuge sindbeschrieben in Abbildung 4. </li><li> Legen Sie Biopsie (etwa 15-50 mg) in Zirkonium Rotor (Abbildung 4 (1)) und füllen den Rest des Volumens mit reinem D 2 O für die NMR-Lock. Achten Sie darauf, keinen Blasen, da diese die Qualität der späteren Shim und die Qualität der Datenerfassung ändern würde. </li><li> Legen Sie 50 ul Teflon spacer (Abbildung 4) (2) mit der Zylinderschraube (Abbildung 4 (5)). Schrauben Sie es und kalibrieren es mit dem Tiefenmesser an der kurzen Seite (Abbildung 4 (8)). Bei diesem Schritt ist es wichtig, ein besonderes Augenmerk auf die Probe zu zahlen, weil ein Teil davon durch den Abstandhalter Loch austreten kann. Ist dies der Fall ist, dann Teil der Biopsie ist zerstört und Gewicht der Probe ist nicht mehr zuverlässig. Es ist daher notwendig, um wieder von vorne beginnen die Vorbereitung der Probe. </li><li> Setzen Sie den thead pin (Abbildung 4) (3) und schrauben Sie ihn vorsichtig mit dem Schraubendreher (Abbildung 4 (6)). Austrocknen Restwasser mit einem Stück Gewebe. </li><li> Setzen Sie die Kappe (Abbildung 4) (4) an derOberseite des Rotors und fügen Sie ihn in den Rotor Packer (Abbildung 4 (6)). Drücken Sie fest, bis die Kappe vorhanden ist. Es sollte keine Leerzeichen zwischen den Rotor und die Kappe gelassen werden. </li><li> Mark die Hälfte der Unterseite des Rotors mit einem schwarzen Filzstift auf optische Spin-Rate-Erkennung ermöglichen. </li><li> Setzen Sie den Rotor im Inneren des NMR-Spektrometers und rotieren beginnen bei 5 kHz. Acquire 1 H-NMR-Spektrum mit CPMG Pulsfolge 13 nach Hersteller-Richtlinien. </li><li> Verwenden α anomeren Glucose Resonanz 5.22 ppm (Dublett), NMR-Spektren zu kalibrieren. </li><li> Um den Rotor entpacken, indem Sie die Kappe mit dem cap-Entferner (Abbildung 4 (9)) fort. Lösen Sie thead Pin und entfernen Teflon spacer mit dem zylindrischen Schraube. Gründlich mit Wasser und Reinigungsmittel. </li></ol> 5. Repräsentative Ergebnisse Gut mikrobielle Aktivität überwacht mit Urin Metabolic Profiling werden. Eine große Anzahl von Urin mikrobiellenCo-Metaboliten erkennbar durch 1 H-NMR sind in der Literatur 7,14-17 beschrieben worden. Diese mikrobielle Co-Metaboliten sind besonders nützlich, um die Besiedlung zu überwachen, da sie einen schnellen und nicht-invasive Methode zur Schätzung, wenn die neu gegründete Ökosystem stabil ist. 5A zeigt deutlich das Aussehen gut mikrobielle Co-Metaboliten über die Kolonisierung. Diese Figur zeigt eine Urin-metabolischen Profils, indem Sie die in Schritt 2 für ein Tier beschrieben, erhalten kolonisiert 20 Tage nach Verfahren in Schritt 1 beschrieben. Dieses Tier nicht scheiden alle indoxyl Sulfat und sehr geringe Mengen an phenylacetylglycine (PAG) und p-Kresol-Sulfat bei der keimfreien Zustand (Tag 0-blau). Als Kolonisierung fortschreitet, diese 3 Marker Protein-Stoffwechsel der Darmflora deutlich erhöhen, um ein Gleichgewicht am Tag 20 (rot) zu erreichen. Dies ist besonders einfach, für eine Gruppe von Tieren, wie dargestellt in Abbildung 5B mit dem PAG-MonitorResonanz. Dieses Diagramm wurde durch Integration der Fläche unter der Resonanzen in grau in Abbildung 5A (δ 7,40-7,43) hervorgehoben, das entspricht einer spezifischen Resonanz (Triplett) der PAG für eine Gruppe von 7 Tieren erhalten. 1 H Hohe Auflösung Magic Angle Spinning (HR MAS) NMR-Spektroskopie ist eine zerstörungsfreie Methode, die eine schnelle und reproduzierbare Akquisitionen von metabolischen Profilen jeglicher Art von Biopsie-18 ermöglicht. In diesem Protokoll haben wir diese leistungsstarke Technik auf eine hepatische metabolische Profil von 2 Mäuse vor (blau) und nach (rot) Kolonisierung (Abbildung 6) zu erhalten. Diese Abbildung zeigt auch die Informationen, die von einem MAS-NMR-basierte metabolische Profil abgeleitet werden können. Zahlreiche Aminosäuren sowie Metaboliten aus energetischen Stoffwechsel, wie Glukose, Glykogen, Laktat, Triglyceride, (D)-3-Hydroxybutyrat und nicotinurate abgeleitet werden können visualisiert werden. Diese Profile enthalten auch Informationen, die zu oxidativem Stress (zB Ascorbinsäure eincid, Glutathion), Nukleotid-Metabolismus (dh Inosin, Uridin) und Methylamin Stoffwechsel (zB Cholin, Trimethylamin-N-Oxid). In diesem Beispiel ist es sehr klar, dass die keimfreie Maus fast kein Glykogen und sehr geringe Mengen an Glucose und Triglyceride wie bisher 7 veröffentlicht Displays. <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="Abbildung 1."/> Abbildung 1. Übersicht über die Kolonisierung Protokoll. Keimfrei und konventionelle Tiere in Käfigen mit Filtern nebeneinander und ihre Würfe werden ausgetauscht, um progressive Kolonisierung von den herkömmlichen Darmflora (1) erlauben ausgestattet untergebracht. Gut mikrobielle Aktivität überwacht wird mittels 1 H-NMR-basierte Metabolic Profiling (2-3). Leberstoffwechsel wird durch 1 H HR-MAS-NMR-basierte Metabolic Profiling (4-5) beurteilt. <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="Abbildung 2."/> Abbildung 2. Maus lebenr Anatomie. Die Leber ist wie die flache Seite der Orgel Gesichter der Tabelle angezeigt. Für reproduzierbare Biopsien, ist es ratsam, immer die Proben an der Mitte des linken Lappens, wie durch die gestrichelte Rechteck angedeutet. <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="Abbildung 3."/> Abbildung 3 1,7 mm-NMR-Kapillar-Kit mit Mikrovolumina Arbeit Key:.. 1: 2,5 mm-NMR-Mikroröhrchen, 2: 1,7 mm-NMR-Kapillare, 3: Kapillar-Adapter, 4: Extraction Stange. <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="Abbildung 4."/> . Abbildung 4 MAS Läufer, Key:. 1: MAS-Rotor, 2: 50 ul Teflon spacer, 3: THEAD polig, 4: Mütze, 5: Zylinderschraube, 6: Schraubendreher, 7: Rotor Verpacker, 8: Tiefenmesser. <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="Abbildung 5."/> Abbildung 5. Evolution der Harnwege metabolischen Profile während colonization. <ol class="ualpha"><li> Zoom auf den aromatischen Bereich des Spektrums zwischen 6,8 bis 7,8 ppm, wo mikrobielle Co-Metaboliten visualisiert werden. 1 H-NMR-Spektren können, wurden von einer einzigen Person am Tag 0 (blau), 4 (grün), 15 (orange) und 20 abgeleitet (red) Post-Kolonialismus. Die Grauzone entspricht der Fläche, die integriert werden, um das Diagramm in B. Key machen war: 1-MeHistamine: 1-Methylhistamin; Indoxyl-S: Indoxyl Sulfat; Sein: Histidin, p-Cresol-S: p-Cresol-Sulfat; PAG : Phenylacetylglycine. </li><li> Durchschnittliche PAG-Konzentration während der Kolonisation (n = 7). Student-t-Test wurde verwendet, um den Unterschied in PAG-Konzentration an verschiedenen Zeitpunkten zu vergleichen: a: p &lt;0,05 im Vergleich zu Tag 0; b: p &lt;0,01 bis Tag 10 verglichen. </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="Abbildung 6."/> Abbildung 6. Typischer 600 MHz 1 H HR-MAS-NMR-Spektren der Leber Biopsien aus keimfrei (blau) und Ex-keimfrei (re abgeleitetd) Mäusen. Bold Protonen sind verantwortlich für die Triglycerid-Resonanz-Key:. 3-HB: 3-Hydroxybutyrat, GSH: reduziertes Glutathion, TGs: Triglyceride, TMAO: Trimethylamin-N-Oxid.

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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>

Wir haben nichts zu offenbaren.

In diesem Protokoll haben wir beschrieben, eine fortschreitende Besiedlung Verfahren in einer offenen Umgebung zur weiteren Untersuchung der Auswirkungen der Darmflora auf den Leberstoffwechsel von 1 H HR-MAS-NMR-Profiling von intakten Biopsie beurteilt. Verschiedene Methoden der Kolonisation sind in der Literatur beschrieben worden. Die häufigsten Methoden, um Tiere mit einer definierten Mikrobiota besiedeln sind orale Gabe oder verunreinigtes Trinkwasser 19,20. Fecal Impfung kann auch verwendet ...

Tabelle der spezifischen Reagenzien und Geräte: <table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> 2,5 mm Mikroröhrchen </td><td> New Era </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 mm Kapillarrohr </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Kapillar-Adapter </td><td> New Era </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> Extraction Stange </td><td> New Era </td><td> NE-341-5 </td><td></td></tr><tr><td> HR-MAS-Rotor BL4 mit 50 ul kugelförmigen Teflon spacer kit </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> Werkzeugsatz für 50 ul-Einsätze </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> Advance-III 600MHz-NMR- </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H HR-MAS-NMR feste Sonde </td><td> Bruker </td><td></td><td></td></tr><tr><td> Deuteriumoxid 99,9% </td><td> Sigma-Aldrich </td><td> 530867-1L </td><td></td></tr><tr><td> 3 - (trimethylsilyl) Propionsäure Säure-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

Es ist bekannt, dass Darmbakterien einen wesentlichen Beitrag an den Host-Homöostase, die eine Reihe von Vorteilen wie Immunschutz und Vitamin-Synthese. Sie liefern auch die Gastgeber mit einer beträchtlichen Menge an Nährstoffen, so dass dieses Ökosystem eine wichtige Stoffwechselorgan. Im Rahmen der zunehmenden Belege für den Zusammenhang zwischen der Darmflora und des metabolischen Syndroms, das Verständnis der metabolischen Interaktion zwischen dem Wirt und seine Darmflora wird immer eine wichtige Herausforderung der modernen Biologie. 1-4 Colonization (auch als Normalisierung bezeichnet) bezeichnet die Einrichtung von Mikroorganismen in einem ehemaligen keimfreie Tier. Es ist zwar ein natürlicher Prozess auftretenden bei der Geburt ist, ist es auch bei erwachsenen keimfreien Tieren verwendet werden, um den Darm floral Ökosystem-Steuerung und weiter zu bestimmen, deren Auswirkungen auf die Host-Stoffwechsel. Ein gängiges Verfahren, um die Kolonisierung Prozesssteuerung ist es, die Sonde Methode mit einem singl verwendene oder eine Mischung von Mikroorganismen. Diese Methode führt zu einer sehr schnellen Kolonisierung und präsentiert den Nachteil, dass sie extrem stressig 5. Es ist daher sinnvoll, um den Stress zu minimieren und eine langsamere Kolonisierung zu erhalten, um schrittweise beobachten die Auswirkungen der bakteriellen Etablierung auf dem Host-Stoffwechsel. In diesem Manuskript, beschreiben wir ein Verfahren zur Änderung des Leberstoffwechsel während einer schrittweisen Kolonisation Verfahren unter Verwendung eines nicht-destruktiven Metabolic Profiling-Technik zu beurteilen. Wir schlagen vor, gut mikrobielle Besiedlung durch die Beurteilung des Darms mikrobielle Stoffwechselaktivität durch die renale Ausscheidung von mikrobiellen Co-Metaboliten durch 1 H-NMR-basierte Metabolic Profiling wider überwachen. Dies ermöglicht eine Aufwertung der Stabilität gut mikrobielle Aktivität über die stabile Etablierung des Darms mikrobiellen Ökosystems in der Regel durch die Überwachung der fäkalen Bakterien durch DGGE (denaturierende Gradienten-Gelelektrophorese) bewertet. 6 DerBesiedlung erfolgt in einem herkömmlichen offenen Umgebung und ist von einem schmutzigen Einstreu verschmutzt durch konventionelle Tiere, die als Kontrollen dienen wird eingeleitet. Nagetiere zu koprophage Tiere, sorgt diese eine homogene Besiedlung wie zuvor beschrieben. 7 Hepatische Metabolic Profiling wird direkt von einer intakten Leberbiopsie mit 1 H Hohe Auflösung MAS-NMR-Spektroskopie gemessen. Diese semi-quantitative Methode bietet einen schnellen Weg, um zu beurteilen, ohne Beschädigung der Zellstruktur, schätzen die Hauptmetaboliten wie Triglyceride, Glucose und Glykogen, um weiter die komplexen Wechselwirkungen zwischen Kolonialisierung und der Leberstoffwechsel 7-10. Diese Methode kann auch für alle Gewebebiopsie 11,12 angewendet werden.

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

Eine progressive Kolonisierung Verfahren wird beschrieben, weitere Beurteilung ihrer Auswirkungen auf den Host Leberstoffwechsel. Colonization wird überwacht nicht-invasiv durch die Auswertung der Ausscheidung von mikrobiellen Co-Metaboliten durch NMR-basierte Metabolic Profiling während Leberstoffwechsel von High Resolution Magic Angle Spinning (HR MAS)-NMR-Profiling von intakten Biopsie beurteilt wird.

Die Beurteilung der Leber metabolischen Veränderungen während Progressive Colonization von Keimfrei Maus durch 1 H-NMR-Spektroskopie

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 Aufrufe.  The University of Reading. Eine progressive Kolonisierung Verfahren wird beschrieben, weitere Beurteilung ihrer Auswirkungen auf den Host Leberstoffwechsel. Colonization wird überwacht nicht-invasiv durch die Auswertung der Ausscheidung von mikrobiellen Co-Metaboliten durch NMR-basierte Metabolic Profiling während Leberstoffwechsel von High Resolution Magic Angle Spinning (HR MAS)-NMR-Profiling von intakten Biopsie beurteilt wird.

Serie: 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.

Die Quantifizierung der Pilzbesiedlung, Sporogenese und Produktion von Mykotoxinen 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 ...

Forschung

Immunologie und Infektion

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.

Colonization von Euprymna scolopes Squid durch 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.

Nachweis von microRNAs in Mikroglia durch Real-time PCR in Normal-und ZNS Während 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.

Charakterisierung von Entzündungsreaktionen während der intranasalen Kolonisation mit 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.

Beurteilung der Anti-Pilz-Aktivität von isolierten Alveolarmakrophagen durch konfokale Mikroskopie

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.

Beurteilung der bakteriellen Invasion von Herzzellen in Kultur und Herzkolonisation bei infizierten Mäusen mit 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.

Selektive Ernte von Marginating-Leber-Leukozyten

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.

Ein Mausmodell der Gruppe B<em&gt; Streptococcus</em&gt; Vaginal Colonization

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.

Molekulare Diffusion in Plasmamembranen von primären Lymphozyten mittels Fluoreszenz-Korrelations-Spektroskopie gemessen

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.

Erkennung von Enterohemorrhagic Escherichia Coli Besiedlung in murinen Host durch nicht-invasive In Vivo Biolumineszenz-System

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