Die Autoren möchten die Unterstützung in bezug auf dieses Manuskript von der Europäischen Kommission Siebten Rahmenprogramms (PIOF-GA-2009 bis 255.466) und dem Irish Health Research Board (HRA_POR/2010/138) quittieren. Lux-markierten E. coli war eine Art Geschenk von Dr. Cormac Gahan, University College Cork.

Ein. Tumorinduktion <ol><li> Für die Routine Tumorinduktion wurde die minimale tumorigenen Dosis von Zellen in 200 ul serumfreiem Kulturmedium suspendiert subkutan (sc) in die Flanke frei von Infektionen 6-8 Wochen alte weibliche Balb / C oder athymischen MF1-nu/nu Mäusen injiziert n = 6 (Harlan, Oxfordshire, UK) (1 x 10 6 4T1-Zellen) mit einer 21-Gauge Injektionsnadel. Die Lebensfähigkeit der Zellen zur Inokulation verwendet wurde, war größer als 95%, wie durch visuelle Zählung mit einem Hämozytometer und Trypan Blue Dye Exclusion (Gibco) bestimmt. </li><li> Nach Tumor-Gründung, wurden Tumoren erlaubt, zu wachsen und zu entwickeln und wurden zweimal wöchentlich überwacht. Das Tumorvolumen wurde gemäß der Formel V = (ab 2) Π / 6, wobei a der längste Durchmesser des Tumors ist und b der längste Durchmesser senkrecht zu a Durchmesser berechnet. </li></ol> 2. Bakterielle Zubereitung <ol><li> Der Bakterienstamm in diesem Protokolls in verwendetenIch war E. coli K-12 MG1655, ein Nicht-Protein-Toxin-exprimierenden Stamm, beherbergen eine luxABCDE-codierenden Plasmid, indem die Bakterien nachgewiesen werden können BLI. E. coli MG1655 enthaltend die integrierte luxABCDE wurde aerob bei 37 ° C in LB-Medium (Sigma-Aldrich, Irland) werden mit 300 ug / ml Erythromycin (Em) ergänzt. Die Biolumineszenz Derivat des MG1655 wurde mit dem Plasmid p16S lux, welche die konstitutive P HELP luxABCDE Operon 1 enthält. </li><li> Für die Zubereitung für die Verabreichung an Mäuse wurden die Kulturen in LB-Medium bei 37 ° C in einem Schüttler bei 200 UpM inkubiert bis zur mittleren logarithmischen Phase (optische Dichte bei 600 nm). Bakterien wurden durch Zentrifugation (6.000 × g für 5 min) gewaschen, mit PBS (Sigma), verdünnt in PBS und 1 × 10 7 koloniebildenden Einheiten (KBE) / ml für intravenöse Verabreichung oder 1 x 10 10 für Schlundsonde. </li></ol> 3. Bakterielle Administrationtion <ol><li> Mäuse wurden randomisiert in Versuchsgruppen eingeteilt, wenn Tumoren erreicht etwa 100 mm 3 im Volumen. Für die intravenöse Verabreichung erhalten zurückhaltende Mäusen je 10 6 Zellen in 100 ul, direkt in die laterale Schwanzvene mit einer 28G Injektionsnadel injiziert. Die Keimzahl jeder Inokulum wurde durch nachträgliche Beschichtung bestimmt. </li><li> Für GIT Kolonisierung Studien wurden 10 9 Bakterienzellen oral in 100 ul pro Maus über eine Sonde verabreicht, an drei aufeinanderfolgenden Tagen. Vorbestehenden kommensalen Bakterien-Spiegel wurden vor dem Zuführen durch Zugabe von 5 mg / ml Streptomycin in Maus Trinkwasser für 7 Tage vor dem Beginn der Schlundsonde 1 verringert. </li></ol> 4. Biolumineszenz-Bildgebung <ol><li> 2D in vivo Bildgebung BLI wurde unter Verwendung des IVIS100 (Bremssattel). Zu definierten Zeitpunkten nach bakteriellen Verwaltung, wurden die Mäuse betäubt mit Caliper die XGI-8 Gas AnästhesieSystem mit 3% Isofluran und Ganzkörper-Bildanalyse in der IVIS 100 für 2-5 min bei hoher Empfindlichkeit durchgeführt. </li><li> Bei 3D-Bildgebung, wurden anästhesiert Mäusen in einem Maus-Bildgebung Shuttle Innenseite des optischen Abbildungssystem zum dorsalen Bildgebung (IVIS Spectrum, Zangenbremse) platziert. Um Bilder des bakteriellen Luciferase Signal für 3D optische Rekonstruktion erwerben wurden Emissionsfilter Wellenlängen im Bereich von 500 bis 580 nm mit bin 16 Erfassungszeiten von 3-4 min pro Filter, um das Signal-Rausch-Verhältnis zu maximieren. Als Teil dieser Abfolge Bildaufnahme wurde ein strukturiertes Licht erhaltenen Bildes, um eine Höhe der Karte zu definieren. Diese Karte eingegeben wurde diffuse Licht bilderzeugende Tomographie (DLIT) Rekonstruktionen Algorithmen, die verwendet werden, um ein 3D Bild unter Verwendung eines optischen nichtnegative kleinsten Quadrate Optimierung 2 zu bilden waren. </li><li> Image Analysis: Regionen von Interesse wurden identifiziert und quantifiziert mit Wohn Image Software (Caliper). </li></ol> 5. VertreterErgebnisse In dieser Studie wurden die nicht-pathogenen kommensalen Bakterien E.coli K-12 MG1655 Exprimieren des luxABCDE Operon war IV Mäusen sc 4T1 Xenograft verabreicht. Bakterielle lux Signal wurde speziell in Tumoren von Mäusen nachgewiesen post IV-Verwaltung (Abbildung 2). Kultur von Bakterien aus Erholung Probe Mäusen validiert die Existenz eines linearen Zusammenhangs zwischen lebensfähigen Bakterienzahl und der Lichtmenge erfasst wird (Abbildung 3). In vivo Bildgebung von oral verabreichten kommensalen Bakterien im GIT wird auch unter Verwendung von 3D BLI. <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="Abbildung 1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> Abbildung 1. Protokoll Timeline. Subkutanen Tumoren in Mäusen induziert und Bakterien auf Tumor-Entwicklung (100 mm 3) verabreicht wird. Live-Mäuse sind BLI imAlter an verschiedenen Zeitpunkten nach bakteriellen Verwaltung (Pfeile zeigen typische mal). <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Verwaltung von E. coli MG1655 luxABCDE um tumortragenden Mäusen. Subkutane 4T1 Tumoren wurden in MF1 nu / nu Mäusen und E. induzierte coli MG1655 luxABCDE auf Tumor-Entwicklung verwaltet. Jedes Tier erhielt 10 6 Zellen direkt in die laterale Schwanzvene injiziert. Die Mäuse wurden zu vier Zeitpunkten während der Studie (schwarze Punkte z-Achse und Bilder) mit anschließender Wiederherstellung lebensfähiger Bakterien (cfu) aus Tumoren von Probe geopfert Mäusen (Balkendiagramm) abgebildet wird. Erhöhung der Keimzahlen und Plasmid-Genexpression spezifisch in Tumoren wurde im Laufe der Zeit (Vertreter Maus pro Zeitpunkt dargestellt) beobachtet. <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</a> .<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Relationship Between Intratumorale Bakterienzahl und Biolumineszenz. Lebensfähigen Bakterien in Tumoren wurden durch ex vivo Bakterienkultur von Tumoren im Anschluss an BLI an verschiedenen Zeitpunkten nach intravenöser Verabreichung aufgezählt. Anmelden Werte Keimzahlen (KBE) in Bezug auf in vivo bioluminesce Einheiten werden grafisch dargestellt. Eine robuste Korrelation zwischen Keimzahlen und bakteriellen Biolumineszenz-Signale beobachtet R 2 = 0,9717 ein. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="Abbildung 4" /> Abbildung 4. 3D IVIS Image Of Murine Gastrointestinal Tract kolonisiert von E. coli MG1655. Das GIT von Mäusen wurde durch orale Verabreichung von 10 9 cfu E. kolonisiert coli an drei aufeinanderfolgenden Tagen. Eine Probe isoliert Bild von 3D-Tomographie des kolonisierten Maus gezeigt.3D-Bilder zeigen eine digitale Maus atlas des Skeletts an anatomische Registrierung ein. E. coli MG1655 Biolumineszenz ist sichtbar in grün bei niedrigeren und lila auf höheren Ebenen.

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Keine Interessenskonflikte erklärt.

Im Rahmen der Gentherapie hat sich die Verwendung von biologischen Agenzien zur Abgabe von therapeutischen Genen für Patienten sehr vielversprechend 3-5 gezeigt. Wie Viren erlauben die angeborenen biologischen Eigenschaften von Bakterien effizienten DNA-Abgabe an Zellen oder Gewebe, insbesondere im Zusammenhang mit Krebserkrankungen. Es hat sich gezeigt, daß Bakterien in der Lage sind natürlich Homing auf Tumoren, wenn systemisch verabreicht, was zu hohen Replikation lokal entweder außerhalb (nicht-invasi...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 4T1-Zelllinie </td><td> ATCC </td><td> CRL-2539 </td><td> Syngenen Brustkrebs-Modell aus einer spontan entstehenden BALB / c Brusttumor abgeleitet </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich </td><td> D6429 </td><td> Dulbecco modifiziertem Eagle-Medium </td></tr><tr><td> PBS </td><td> Sigma-Aldrich </td><td> D8537 </td><td> Phosphate Buffered Saline </td></tr><tr><td> Xenogen IVIS </td><td> Caliper Life Sciences </td><td></td><td> IVIS 100 für 2D-Bildgebung; IVIS Spectrum für 3D. </td></tr><tr><td> Luria Broth Miller (LB) </td><td> Sigma-Aldrich </td><td> L2542 </td><td> Wachstumsmedium für E. coli </td></tr&gt; <tr><td> Erythromycin </td><td> Sigma-Aldrich </td><td> E5389 </td><td> Antibiotikum </td></tr><tr><td> Streptomycin </td><td> Sigma-Aldrich </td><td> S9137 </td><td> Antibiotikum </td></tr><tr><td> MF1nu/nu Mäusen </td><td> Harlan (UK) </td><td> 069 (nu) / 070 (nu / +) </td><td> Hsd: athymischer-Foxn1nu </td></tr><tr><td> Balb / c-Mäusen </td><td> Harlan (UK) </td><td> 066 </td><td> Haplotyp: H-2 d </td></tr><tr><td> Gavage Nadel </td><td> Vet-Tech-Solutions (UK) </td><td> DE009 </td><td> 22G x 38mm gerade Magensonde Nadel </td></tr><tr><td> Spritze für iv Injektion </td><td> BD Biosciences </td><td> 309.309 bis 1 ml </td><td> Insulinspritze mit 28 G x ½ Zoll mikro-feinen IV Nadel. </td></tr><tr><td> Spritze für Tumorinokulation </td><td> Braun </td><td> 9161376V </td><td> Omnifix 26 G x &amp;frac12; Zoll-Nadel </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

Dieses Video beschreibt den Einsatz des ganzen Körpers bioluminesce Imaging (BLI) für die Untersuchung von bakteriellen Handel mit lebenden Mäusen, mit einem Schwerpunkt auf der Verwendung von Bakterien in der Gen-und Zelltherapie bei Krebs. Bakterien stellen eine attraktive Klasse von Vektor für die Krebstherapie, die über eine natürliche Fähigkeit, vorzugsweise wachsen innerhalb Tumoren nach systemischer Verabreichung. Bakterien entwickelt, um die lux Genkassette Erlaubnis BLI Nachweis der Bakterien und gleichzeitig Tumorstellen auszudrücken. Die Lage und Höhe der Bakterien innerhalb Tumoren im Laufe der Zeit leicht geprüft werden kann, visualisiert in zwei oder drei Dimensionen. Das Verfahren ist für ein breites Spektrum von Bakterien-Spezies und Tumorxenotransplantat Typen. Dieser Artikel beschreibt das Protokoll für die Analyse der Biolumineszenz Bakterien im subkutanen Tumor tragenden Mäusen. Visualisierung von symbiotischer Bakterien im Magen-Darm-Trakt (GIT) von BLI wird ebenfalls beschrieben. Diese leistungsstarke und billig, Bildgebung in Echtzeit Strategie Vertreterts eine ideale Methode zur Untersuchung von Bakterien in vivo im Kontext der Krebsforschung, insbesondere Gentherapie, und Infektionskrankheiten. Dieses Video beschreibt das Verfahren für die Untersuchung lux-markierten E. coli in lebenden Mäusen, zeigt die räumliche und zeitliche Auslesen erreichbaren Nutzung BLI mit dem IVIS System.

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

Dieser Artikel beschreibt die Verwaltung von<em&gt; Lux-getaggten</em&gt; Bakterien an Mäusen und anschließende<em&gt; In vivo</em&gt; Analyse mit IVIS Biolumineszenz-Bildgebung.

Bioluminescent Bakterielle Imaging<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K Aufrufe.  University College Cork. Dieser Artikel beschreibt die Verwaltung von Lux-getaggten Bakterien an Mäusen und anschließende In vivo Analyse mit IVIS Biolumineszenz-Bildgebung.

Serie: Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

In-vivo-Imaging von transgenen Leishmania Parasiten in einer Live-Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Forschung

Immunologie und Infektion

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Nicht-invasive Bildgebung von Leukozyten Homing und Migration In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Die Beurteilung stomatäre Response to Bakterienzellen Live mit Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Mit Bioluminescent Imaging, um Synergie von Untersuchen Streptococcus pneumoniae Und Influenza A Virus in Infant Mäuse

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminescent &#39;Reporter Phage für den Nachweis von Kategorie A bakteriellen Erregern

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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 ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In-vivo-Imaging-Methode zu unterscheiden, akuten und chronischen Entzündungen

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation und Multi-phänotypischen High-Content-Screening<em&gt; Coxiella burnetii</em&gt; Transposon-Mutanten

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In-vitro- und In-vivo-Modell, um die bakterielle Adhäsion an der Gefäßwand unter Strömungsbedingungen Studieren

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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