Diese Arbeit wurde vom NIH RO1 AI079253 und Department of Veterans Affairs finanziert.

Ein. Tiermodelle <ol><li> Mäuse: Verwenden p47 phox-/ - Mäusen und Alter und Geschlecht abgestimmt C57BL6/DBA Mäusen. Holen Sie die Genehmigung für die Experimente von Institutional Animal Care und Use Committee. </li><li> Anästhesie: Verwenden Sie eine kontinuierliche Isofluran Verwaltungssystem der Narkose zu induzieren. Der Verdampfer System (VetEquip) mit Isofluran (2-3%) gefüllt. Bestätigen Sie, dass Mäuse vollständig betäubt durch die Beobachtung der Atmung, Bewegung und Cornealreflex in Reaktion auf äußere Reize sind. </li><li> Chirurgische Verfahren: Reiben Sie die OP-Bereich (Tischgerät) mit 70% Ethanol. Trage sterile Handschuhe und einen sauberen OP-Kittel und Maske. Bereiten Sie die Mäuse durch Clipping die Haare und Aufbringen betadine abwechselnd mit 70% igem Ethanol zu dem Bereich, wo der Schnitt gemacht werden soll. Durchführung von Operationen auf einer sterilen Oberfläche und mit sterilen Instrumenten. Monitor Mäusen post-operativ, bis wach und frei zu bewegen. </li><li> Intratracheale Zymosan Verwaltung <ol><li> Verwalten Anästhesiewie in 1.2 beschrieben. </li><li> Desinfizieren Operationsgebiet (Hals) mit Betadin und 70% Ethanol und setzen die Luftröhre durch chirurgische Dissektion. </li><li> Pierce Trachea mit einer 27-Gauge-Nadel und injizieren Zymosan (St. Louis, MO) in einer Dosis von 1 pg / g unter Verwendung einer 0,5 ug / ul Lösung (Gesamtvolumen 50 pl für eine Maus von 25 g) in sterilem PBS aufgelöst. </li><li> Schließen Mäusen Hals gewickelt mit 5-0 sterile Seidennähte unter aseptischen Bedingungen. </li><li> Bild nach 4 Stunden und 24 Stunden. </li></ol></li><li> Blinddarm Ligatur und Punktion <ol><li> Verwalten Anästhesie in 1.2 beschrieben. </li><li> Clip Haar in der OP-Bereich (Abdomen) und desinfizieren mit betadine und 70% Ethanol. </li><li> Führen Sie eine Mittellinie Laparotomie und Identifizierung der Blinddarm. </li><li> Ligieren das distale 50% der exponierten Caecum mit 4-0 Seidenfaden und Durchlöcherung Caecum distal zur Ligation mit einem Durchgang von einer 21-Gauge-Nadel. </li><li> Schließen Sie den Schnitt mit 4-0 sterile Seidenfäden. </li><li> Bild nach 4 h und24 Std. </li></ol></li><li> Oral Tetrachlorkohlenstoff (CCl 4) Verwaltung <ol><li> Verwalten Anästhesie in 1.2 beschrieben. </li><li> Verwalten CCl 4 (2 pg / g, St. Louis, MO) in Maisöl durch orale Gabe aufgelöst. Das Verfahren der CCl4 Verwaltung sollte in einem bestimmten Bereich in Übereinstimmung mit IBC / EHS-Richtlinien durchgeführt werden. </li><li> Bild nach 4 Stunden und 24 Stunden. </li></ol></li></ol> 2. Der Erwerb einer Bioluminescent Bild <ol><li> Initialisieren Sie die IVIS 200 (Xenogen Corporation, Alameda, CA) Imaging System, auf Lumineszenz-Modus und warten, bis die Charge-Coupled Device (CCD) Temperatur zu sperren. </li><li> Wählen Belichtungszeit, Binning (mittel) und F / Stop (8) Einstellungen. </li><li> Ort Mäusen in Rückenlage in der Bildgebung Kammer auf einem Heiztisch zur normalen Körpertemperatur aufrecht zu erhalten. Verwalten Anästhesie in 1,2 via Nasenkonus beschrieben, während Mäuse in der IVIS Bildgebung Kammer sind. </li><li> Verwalten L-012<html> (20 g / g) in sterilem PBS (10 ug / ul) intravenös über retroorbitalen Einspritzung zu jedem Zeitpunkt zum Abbilden ausgewählt gelöst. </li><li> Aufnehmen von Bildern beginnen bei 2 min nach L-012-Injektion. Wir verwenden in der Regel eine 40 sec Belichtung. </li></ol> 3. Data Analysis mit Region of Interest <ol><li> Analysieren von Daten mit Wohn Image Software v.4.2 (Xenogen Corporation, Alameda, CA). </li><li> Öffnen Sie ein Bild und wählen Sie Messung der Region of Interest-Tools. </li><li> Wählen der Region of Interest Gestalt und Größe auf der Brust und / oder Bauch nach Überlagern eines Pseudo-farbigen digitalen Bild (repräsentiert Photonendetektion) über einem fotografischen Abbild der Maus. </li><li> Quantifizierung Signalintensität (Photonenfluss) aus der Region of Interest. </li></ol> 4. Statistiken <ol><li> Statistische Analysen verwenden Softwarepaket Zwei-Wege-ANOVA mit Bonferroni post-Tests durchführen zu einzelnen Zeitpunkten. </li></ol>tle &quot;&gt; 5. Repräsentative Ergebnisse Zymosan ist eine proinflammatorische Hefezellwand Produkt und ein potenter Aktivator der NADPH-Oxidase-3. Wir haben bereits gezeigt, dass intratracheale Zymosan induziert eine robustere neutrophilen Lungenentzündung und pro-inflammatorische Zytokin-Produktion in p47 phox-/ - im Vergleich zu Wildtyp-Mäusen 1. Hier war unser Ziel, ROS-Generation in den Lungen von Wildtyp-und p47 phox-/ compare - Mäusen nach Zymosan Herausforderung. Bei 4 Stunden nach Zymosan intratracheale Verabreichung, beobachteten wir eine deutliche Erhöhung der Photonenemission über der Brust in Wildtyp-Mäusen im Vergleich zur Basislinie sowie einer Erhöhung der Photonenemission in Wildtyp p47 phox gegenüber-/ - Mäusen bei 4 h und 24 h. Im Gegensatz dazu Biolumineszenz Signale in p47 phox-/ - blieben Mäuse Ausgangswert bei 4 Stunden nach Zymosan Behandlung (Abb.1 AB). Somit scheint NADPH-Oxidase zu be die Hauptquelle der ROS-Generation in der Lunge nach Zymosan Verwaltung. Weiter untersuchten wir ROS-Produktion in der CLP-induzierten Sepsis-Modell. Sepsis ist eine lebensbedrohliche Syndrom mit end-Organverletzung verbunden sind, einschließlich akutem Lungenversagen (ALI) und akutes Atemnotsyndrom (ARDS) 4, 5. ROS-vermittelten Verletzungen wurde als ein wichtiger Faktor für Sepsis-induzierten Multi-Organ-Dysfunktion. Wir fanden, dass ROS signifikant wurden über der Brust (4 h) und über den Bauch (24 h) nach CLP in Wildtyp-Mäusen im Vergleich zum Ausgangswert erhöht. Allerdings ROS Ebenen in p47 phox-/ - waren Mäuse ähnlich den Ausgangswerten zu beiden Zeitpunkten (Abb.2 AC). Diese Ergebnisse zeigen, dass ROS Generation in der CLP-induzierten Sepsis Mausmodell NADPH-Oxidase-abhängig ist. Schließlich haben wir Biolumineszenz Bildgebung ROS-Produktion in einer CCl 4-induzierten Leberschäden Modus erkennenl. CCl 4 bewirkt hepatozellulären Nekrose, und wurde verwendet, um sowohl akute Leberschädigung und Leberfibrose 6 modellieren. Im Gegensatz zu den zuvor beschriebenen Modelle von Entzündungen und Verletzungen, fanden wir, dass ROS-Produktion in den Bauch bescheiden war (etwa 35%) erhöht gegenüber dem Ausgangswert in p47 phox-/ - Mäusen bei 4 h nach CCl 4 Verwaltung. Jedoch war das Ausmaß der CCl 4-induzierten ROS signifikant größer in Wildtyp-Mäusen als in p47 phox-/ - Mäusen (Abb.3 AB). Diese Daten legen nahe, dass sowohl p47 phox-haltigen NADPH-Oxidase-abhängige und-unabhängige ROS Generation in akuten CCl 4-induzierten Leberschäden auftreten. <img alt="Abbildung 1" src="/files/ftp_upload/3925/3925fig1.jpg" /> Abbildung 1. ROS-Produktion in der Lunge nach Zymosan Behandlung. A) Repräsentative bioluminescence Bildgebung von ROS-Produktion. Beachten Sie, dass zusätzlich zu der Brust, Lumineszenz detektierbar über den Bauch sowohl Wildtyp und p47 phox-/ ist - Mäusen und abdominalen Lumineszenz ist unverändert durch Zymosan Behandlung. B) Photonen von der Brust von Wildtyp-und p47 phox-/ - Mäusen nach einer einzigen intratracheale (IT) Injektion von Zymosan. Biolumineszenz-Bildgebung wurde an der Grundlinie, 4 h und 24 h durchgeführt. Die Lichtemission aus der Region von Interesse über die Brust wurde unter Verwendung des angezeigten Pseudo-Farbskala. Die Ergebnisse werden als Mittelwert ± SE vorgestellt, n = 6-9 pro Gruppe, * = p &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig1large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung</a> . <img alt="Abbildung 2" src="/files/ftp_upload/3925/3925fig2.jpg" /> Abbildung 2. ROS Nachweis nach CLP. A) Repräsentative Biolumineszenz-Bildgebung der ROS-Produktion, B) Photonen aus der Brust, und C) Photonen aus dem Bauch von Wildtyp-und p47 phox-/ - Mäusen zu Studienbeginn, 4 h und 24 h nach CLP. Die Ergebnisse werden als Mittelwert dargestellt ± SE; n = 4-5 pro Gruppe, * = p &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig2large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung</a> . <img alt="Abbildung 3" src="/files/ftp_upload/3925/3925fig3.jpg" /> Abbildung 3. ROS-Produktion in Bauch nach CCl 4-induzierten Leberschäden. A) Repräsentative Biolumineszenz-Bildgebung der ROS-Produktion und B) Photonen aus dem Bauch von Wildtyp-und p47 phox-/ - Mäusen zu Studienbeginn, 4 Stunden und 24 Stunden nach der oralen CCl 4 Behandlung. . N = 4-5 pro Gruppe, * = p &lt;0,05; Ergebnisse werden als Mittelwert ± SE präsentiert <a href="http://www.jove.com/files/ftp_upload/3925/3925fig3large.jpg" target="_blank">Klicken Sie hier für eine größere Zahl</a> </a&gt;.

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Keine Interessenskonflikte erklärt.

&quot;Real-time&quot;-Messung von reaktiven Sauerstoffspezies (ROS) in lebenden Tieren kann durch Fluoreszenz-und Chemilumineszenz-Sonden erreicht werden. Während fluoreszierenden Sonden aus mit schwacher Signal-zu-Rausch-Verhältnisse 12 leiden, ist die beschriebene Aufnahmetechnik empfindlicher für die Detektion der Lichtemission im Anschluss an eine chemische Reaktion mit dem ROS Luminol-basierten Substrat L-012. Wie alle Biolumineszenz Bildgebung, wird diese Methodik durch wellenlängenabhängige Licht ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalognummer </td><td> Kommentare </td></tr><tr><td> L-012 </td><td> Wako Chemicals USA, Inc. </td><td> 120-04891 </td><td></td></tr><tr><td> Zymosan </td><td> Sigma, St. Louis, MO </td><td> Z4250 </td><td></td></tr><tr><td> Tetrachlorkohlenstoff </td><td> Sigma, St. Louis, MO </td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPH-Oxidase ist ein kritischer Enzym, das antibakterielle und antimykotische Wirts vermittelt. Zusätzlich zu seiner Rolle in der antimikrobiellen Abwehrmechanismen hat NADPH-Oxidase kritischen Meldefunktionen, die die Entzündungsreaktion 1 modulieren. Somit ist die Entwicklung einer Methode, um in &quot;real-time&quot; die Kinetik der NADPH-Oxidase-derived ROS-Generation messen erwartet ein wertvolles Forschungsinstrument Mechanismen relevant Verteidigung, Entzündungen und Verletzungen Gastgeber verstehen. Chronische Granulomatose (CGD) ist eine erbliche Erkrankung des NADPH-Oxidase durch schwere Infektionen und übermäßiger Entzündung aus. Aktivierung der Phagozyten NADPH-Oxidase erfordert Translokation seiner cytosolischen Untereinheiten (p47 phox, p67 phox und p40 phox) und Rac an einen membrangebundenen Flavocytochrom (bestehend aus einer gp91 phox und p22 phox Heterodimer). VerlustFunktionsverlust-Mutationen in einem dieser NADPH-Oxidase Komponenten ergeben CGD. Ähnlich wie bei Patienten mit CGD haben gp91 phox-defizienten Mäusen und p47 phox-defizienten Mäusen defekte Phagozyt NADPH-Oxidase-Aktivität und beeinträchtigter Wirtsabwehr 13, 14. Neben Phagozyten, die die NADPH-Oxidase oben beschriebenen Komponenten enthalten, exprimieren eine Vielzahl von anderen Zelltypen unterschiedliche Isoformen von NADPH-Oxidase. Hier beschreiben wir eine Methode zur ROS-Produktion in lebenden Mäusen zu quantifizieren und den Beitrag der NADPH-Oxidase zu ROS-Generation in den Modellen von Entzündungen und Verletzungen abzugrenzen. Dieses Verfahren basiert auf ROS Umsetzung mit L-012 (ein Analogon von Luminol) zur Lumineszenz, die durch eine Charge-Coupled Device (CCD) aufgezeichnet wird basierend emittieren. In der ursprünglichen Beschreibung der L-012-Sonde wurde L-012-abhängigen Chemolumineszenz vollständig von Superoxiddismutase aufgehoben, was anzeigt, dass der Haupt-ROS in dieser Reaktion nachgewiesen war superOxidanion 15. Spätere Studien haben gezeigt, dass L-012 können andere freie Radikale, einschließlich reaktiven Stickstoffspezies 16, 17 zu detektieren. Kielland et al. 17 zeigte, dass die topische Anwendung von Phorbolmyristatacetat, ein potenter Aktivator der NADPH-Oxidase, führte zu Oxidase-abhängigen ROS Generation, die in Mäusen unter Verwendung des lumineszierenden Sonde L-012 detektiert werden könnte NADPH. In diesem Modell zeigten sie, dass L-012-abhängigen Lumineszenz in p47 phox-defizienten Mäusen wurde aufgehoben. Wir verglichen ROS-Generation in Wildtyp-Mäusen und NADPH-Oxidase-Mangel p47 phox-/ - Mäusen 2 in den folgenden drei Modelle: 1) intratracheale Verabreichung von Zymosan, eine pro-inflammatorische Pilzzellwand abgeleiteten Produkt, das NADPH-Oxidase aktivieren kann, 2) cecal Ligatur und Punktion (CLP), ein Modell des intra-abdominalen Sepsis mit sekundärer akuter Lungenentzündung und Verletzungen, und 3) mündliche Tetrachlorkohlenstoff(CCl 4), ein Modell der ROS-abhängige Leberschädigung. Diese Modelle wurden speziell ausgewählt, um NADPH-Oxidase-abhängigen ROS-Generation im Rahmen der nicht-infektiösen Entzündungen, polymikrobiellen Sepsis und toxininduzierte Organverletzung auszuwerten sind. Vergleicht man Biolumineszenz in Wildtyp-Mäusen p47 phox-/ - Mäusen ermöglicht es uns, den spezifischen Beitrag von ROS durch p47 phox-haltigen NADPH-Oxidase der Biolumineszenz-Signal in diesen Modellen generiert abzugrenzen. Biolumineszenz-Bildgebung Ergebnisse, die eine erhöhte ROS Ebenen in Wildtyp-Mäusen im Vergleich zu p47 nachgewiesen phox-/ - Mäusen zeigten, dass NADPH-Oxidase die Hauptquelle der ROS-Generation ist in Reaktion auf entzündliche Reize. Diese Methode bietet eine minimal-invasive Ansatz für &quot;real-time&quot; Überwachung der ROS-Generation während der Entzündung in vivo.

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH-Oxidase ist die wichtigste Quelle von reaktiven Sauerstoffspezies (ROS) in Phagozyten. Aufgrund der Kurzlebigkeit von ROS, ist es schwierig zu messen und zu überwachen ROS Ebenen in lebenden Tieren. Ein minimal invasives Verfahren zur seriellen Quantifizierung von ROS in lebenden Mäusen beschrieben.

Biolumineszenz Imaging von NADPH-Oxidase-Aktivität in verschiedenen Tiermodellen

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

bioluminescence-imaging-nadph-oxidase-activity-different-animal

Research

JoVE Journal

Immunology and Infection

16.1K Aufrufe.  Vanderbilt University School of Medicine. NADPH-Oxidase ist die wichtigste Quelle von reaktiven Sauerstoffspezies (ROS) in Phagozyten. Aufgrund der Kurzlebigkeit von ROS, ist es schwierig zu messen und zu überwachen ROS Ebenen in lebenden Tieren. Ein minimal invasives Verfahren zur seriellen Quantifizierung von ROS in lebenden Mäusen beschrieben.

Serie: Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Diagnose von Ekto-und Endoparasiten in Laborratten und Labormäusen

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Forschung

Immunologie und Infektion

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Speichel, Speicheldrüse, und Hämolymphe Abholung Ixodes scapularis Zecken

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Langzeitintravital Multiphotonen Mikroskopie Imaging von Immunzellen in gesunden und kranken Leber Mit CXCR6.Gfp Reporter Mäuse

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Isolation und Transplantation von Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Biolumineszenz-Bildgebung zur Erkennung von Late Stage-Infektion der afrikanischen Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Durchflusszytometrische Analyse von natürlichen Killerzellen lytische Aktivität in menschlichem Vollblut

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

In - vivo - Untersuchung von Antimicrobial Blau Lichttherapie für Multiresistente Acinetobacter baumannii Brennen Infektionen Biolumineszenz - Bildgebung

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Nicht-invasiv<em&gt; In vivo</em&gt; Fluoreszenzoptische Bildgebung der entzündlichen MMP-Aktivität unter Verwendung eines aktivierbaren Fluoreszenzbildgebers

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Live-Imaging von Anti-Pilz-Aktivität von Human Primary Neutrophilen und Monozyten in Reaktion auf<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

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 ...