Les auteurs tiennent à remercier un soutien pertinent à ce manuscrit de la Commission européenne du septième programme-cadre (PIOF-GA-2009-255466) et le Conseil de la santé irlandais de recherche (HRA_POR/2010/138). Lux-étiqueté E. coli était un cadeau aimable de M. Cormac Gahan, University College Cork.

1. L&#39;induction des tumeurs <ol><li> Pour l&#39;induction de tumeurs de routine, la dose tumorigène minimale de cellules en suspension dans 200 ul de milieu de culture sans sérum a été injecté par voie sous cutanée (sc) dans le flanc de l&#39;infection gratuit 6-8 semaines vieilles femelles Balb / C ou athymiques souris MF1-nu/nu n = 6 (Harlan, Oxfordshire, Royaume-Uni) (1 x 10 6 cellules 4T1) en utilisant une aiguille de seringue de calibre 21. La viabilité des cellules utilisées pour l&#39;inoculation était supérieure à 95% telle que déterminée par un comptage visuel en utilisant un hémocytomètre et d&#39;exclusion du bleu de trypan (Gibco). </li><li> Après la création de la tumeur, les tumeurs ont été autorisés à se développer et ont été suivis deux fois par semaine. Le volume tumoral est calculé selon la formule V = (ab 2) Π / 6, où a est le plus grand diamètre de la tumeur et b est le diamètre le plus long perpendiculaire à un diamètre. </li></ol> 2. Préparation bactérienne <ol><li> La souche bactérienne utilisée dans le présent ProtocoJ&#39;étais E. coli K-12 MG1655, une protéine non-toxine-exprimant la souche, hébergeant un plasmide codant pour luxABCDE qui permet aux bactéries à détecter par BLI. E. coli MG1655 contenant le luxABCDE intégré a été cultivé en aérobiose à 37 ° C en milieu LB (Sigma-Aldrich, Irlande) additionné de 300 ug / ml d&#39;érythromycine (Em). Le dérivé de bioluminescence MG1655 a été créé en utilisant le plasmide p16S lux qui contient le P AIDE constitutive luxABCDE opéron 1. </li><li> Pour la préparation pour l&#39;administration à des souris, les cultures ont été incubées dans du milieu LB à 37 ° C dans un agitateur à 200 tours par minute à la phase mi-log (densité optique à 600 nm). Les bactéries ont été récoltées par centrifugation (6000 xg pendant 5 min), lavées avec du PBS (Sigma), et dilué dans du PBS 1 × 10 7 unités formant colonies (UFC) / ml pour administration par voie intraveineuse, ou 1 x 10 10 pour gavage. </li></ol> 3. Bactérienne Administrationtion <ol><li> Les souris ont été divisés au hasard en groupes expérimentaux lorsque les tumeurs ont atteint environ 100 mm 3 de volume. Pour l&#39;administration par voie intraveineuse, les souris ont reçu chacun sobres 10 6 cellules dans 100 ul, injecté directement dans la veine latérale de la queue en utilisant une aiguille de seringue 28G. Le nombre viable de chaque inoculum a été déterminé par placage rétrospective. </li><li> Pour les études de colonisation GIT, 10 9 cellules bactériennes ont été administrés par voie orale dans 100 pi par souris par gavage, pendant trois jours consécutifs. Préexistantes commensales niveaux bactériens ont diminué avant de nourrir par addition de 5 mg / ml de streptomycine dans l&#39;eau potable de la souris pendant 7 jours avant le début du gavage oral 1. </li></ol> 4. Imagerie par bioluminescence <ol><li> 2D imagerie in vivo BLI a été réalisée en utilisant la IVIS100 (étrier). Au moment après l&#39;administration défini des points bactérienne, souris ont été anesthésiées avec étrier de XGI-8 anesthésie au gazSystème avec isofluorane 3%, et l&#39;ensemble du corps d&#39;analyse d&#39;images a été réalisée dans le IVIS 100 système pendant 2-5 min à haute sensibilité. </li><li> Pour l&#39;imagerie 3D, souris anesthésiées ont été placés dans une navette imagerie souris à l&#39;intérieur du système d&#39;imagerie optique pour l&#39;imagerie dorsale (IVIS spectre, étrier). D&#39;acquérir des images de la luciférase bactérienne signal de la reconstruction 3D optique, des longueurs d&#39;onde d&#39;émission de filtre allant de 500 à 580 nm ont été utilisés avec des temps d&#39;acquisition de bacs de 16 min 3-4 par filtre pour maximiser le rapport signal sur bruit. Dans le cadre de cette séquence d&#39;acquisition d&#39;images, une image de la lumière structurée a été obtenu pour définir une carte de hauteur. Cette carte était entrée diffuse la lumière d&#39;imagerie tomographie (DLit) algorithmes de reconstructions qui ont été utilisés pour former une image 3D optique en utilisant un non-négatif au optimisation carrés 2. </li><li> Analyse d&#39;images: Régions d&#39;intérêt ont été identifiés et quantifiés à l&#39;aide du logiciel Image habitable (étrier). </li></ol> 5. ReprésentantRésultats Dans cette étude, les bactéries commensales non pathogènes E. coli K-12 MG1655 exprimant l&#39;opéron luxABCDE était IV administré à des souris porteuses de xénogreffes de tumeurs sc 4T1. Lux bactérien signal a été détectée spécifiquement dans les tumeurs des souris après administration IV-(Figure 2). Récupération de la culture de bactéries à partir d&#39;échantillons souris valide l&#39;existence d&#39;une relation linéaire entre le nombre de bactéries viables et la quantité de lumière détectée (figure 3). L&#39;imagerie in vivo du oralement administrés par des bactéries commensales dans le GIT est également réalisée en utilisant la 3D BLI. <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="Figure 1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> Figure 1. Protocole Timeline. Tumeurs sous-cutanées sont induits chez la souris, et les bactéries administré sur le développement des tumeurs (100 mm 3). Des souris vivantes sont BLI imâgés de moins après l&#39;administration de divers points temporels bactérienne (flèches afficher les temps typiques). <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="Figure 2" /> Figure 2. L&#39;administration de E. coli MG1655 luxABCDE à des souris porteuses de tumeurs. tumeurs sous-cutanées 4T1 ont été induites chez MF1 souris nu / nu et E. coli MG1655 luxABCDE administré sur le développement des tumeurs. Chaque animal a reçu 10 6 cellules injectées directement dans la veine latérale de la queue. Les souris ont été imagées moins quatre points dans le temps au cours de l&#39;étude (points noirs axe z et images) avec récupération ultérieure des bactéries viables (UFC) à partir d&#39;échantillons de tumeurs de souris sacrifiées (graphique à barres). Augmentation du nombre de bactéries et de l&#39;expression du gène plasmidique spécifiquement dans les tumeurs a été observée au cours du temps (souris représentant illustré par point de temps). <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> .<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="Figure 3" /> Figure 3. Relation entre le nombre de bactéries intra-tumorale et la bioluminescence. Bactéries viables dans les tumeurs ont été dénombrés par ex vivo de tumeurs culture bactérienne à la suite de BLI à divers points de temps après l&#39;administration IV. Les valeurs de log du nombre de bactéries (ufc) par rapport aux unités bioluminesce in vivo sont représentées graphiquement. Une forte corrélation entre le nombre de bactéries et de signaux de bioluminescence bactérienne est observée R 2 = 0,9717 1. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="Figure 4" /> Figure 4. 3D Image De IVIS tractus gastro-intestinal murin colonisés par E. coli MG1655. Le GIT de souris a été colonisée par l&#39;administration orale de 10 9 cfu de E. coli pendant trois jours consécutifs. Une image d&#39;échantillon isolé à partir de la tomographie 3D de la souris colonisé est affiché.Images 3D montrent un atlas numérique de la souris sur le squelette de fournir l&#39;enregistrement anatomique. E. coli MG1655 bioluminescence est visible en vert en bas, et de pourpre à des niveaux supérieurs.

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Aucun conflit d&#39;intérêt déclaré.

Dans le cadre de la thérapie génique, l&#39;utilisation d&#39;agents biologiques pour la livraison de gènes thérapeutiques aux patients a montré des résultats prometteurs 3-5. Comme les virus, les propriétés biologiques innés de bactéries permettre la délivrance d&#39;ADN efficace de cellules ou de tissus, en particulier dans le cadre du cancer. Il a été montré que les bactéries sont naturellement capables de ralliement pour des tumeurs lorsqu&#39;elles sont administrés par voie systémique r?...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Lignée cellulaire 4T1 </td><td> ATCC </td><td> CRL-2539 </td><td> Syngénique modèle du cancer du sein dérivée d&#39;une façon spontanée résultant BALB / c tumeur mammaire </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich </td><td> D6429 </td><td> Eagle modifié par Dulbecco à moyen </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 pour l&#39;imagerie 2D; Spectre IVIS pour la 3D. </td></tr><tr><td> Luria Broth Miller (LB) </td><td> Sigma-Aldrich </td><td> L2542 </td><td> Milieu de croissance pour E. coli </td></tr&gt; <tr><td> Érythromycine </td><td> Sigma-Aldrich </td><td> E5389 </td><td> Antibiotique </td></tr><tr><td> Streptomycine </td><td> Sigma-Aldrich </td><td> S9137 </td><td> Antibiotique </td></tr><tr><td> Souris MF1nu/nu </td><td> Harlan (Royaume-Uni) </td><td> 069 (nu) / 070 (nu / +) </td><td> Hsd: athymique Nu-Foxn1nu </td></tr><tr><td> Souris Balb / c </td><td> Harlan (Royaume-Uni) </td><td> 066 </td><td> Haplotype H-2 d </td></tr><tr><td> Aiguille de gavage </td><td> Vet-Tech Solutions (UK) </td><td> DE009 </td><td> 22G x 38mm droite aiguille de gavage </td></tr><tr><td> Seringue pour injection IV </td><td> BD Biosciences </td><td> 309309 à 1 ml </td><td> Seringue à insuline avec 28 x G ½ pouces micro-aiguille fine IV. </td></tr><tr><td> Seringue pour inoculation de la tumeur </td><td> Braun </td><td> 9161376V </td><td> Omnifix 26 x G &amp;frac12; aiguille pouces </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

Cette vidéo décrit l&#39;utilisation de l&#39;imagerie du corps entier bioluminesce (BLI) pour l&#39;étude de la traite des bactéries chez des souris vivantes, avec un accent sur l&#39;utilisation de bactéries en thérapie génique et cellulaire du cancer. Les bactéries présentes une classe intéressante de vecteur pour la thérapie du cancer, qui possède une capacité naturelle à se développer de manière préférentielle dans les tumeurs après administration systémique. Bactéries génétiquement pour exprimer le gène lux cassette d&#39;autorisation BLI détection des bactéries et en même temps les sites tumoraux. L&#39;emplacement et les niveaux de bactéries au sein des tumeurs au fil du temps peut être facilement examinés, visualisées en deux ou trois dimensions. La méthode est applicable à une large gamme d&#39;espèces bactériennes et les types de xénogreffes de tumeurs. Cet article décrit le protocole d&#39;analyse des bactéries bioluminescentes dans les souris porteuses de tumeurs sous-cutanées. Visualisation des bactéries commensales dans le tractus gastro-intestinal (TGI) par BLI est également décrit. Ce puissant et pas cher, en temps réel représentation stratégie d&#39;imageriets une méthode idéale pour l&#39;étude des bactéries in vivo dans le cadre de la recherche sur le cancer, en particulier la thérapie génique et les maladies infectieuses. Cette vidéo décrit la procédure pour l&#39;étude de lux-étiqueté E. coli dans des souris vivantes, ce qui démontre la lecture spatiale et temporelle BLI réalisables en utilisant le système IVIS.

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

Bioluminescent Bacterial Imaging In Vivo

Cet article décrit l&#39;administration de<em&gt; Lux-étiqueté</em&gt; Les bactéries à des souris et ultérieures<em&gt; In vivo</em&gt; Analyse en utilisant l&#39;imagerie par bioluminescence IVIS.

Bioluminescent imagerie bactérienne<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 vues.  University College Cork. Cet article décrit l'administration de Lux-étiqueté Les bactéries à des souris et ultérieures In vivo Analyse en utilisant l'imagerie par bioluminescence IVIS.

Vidéo: 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.

L&#39;imagerie in vivo des parasites Leishmania transgéniques dans un hôte en direct

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

Recherche

Immunologie et infection

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.

L&#39;imagerie non invasive des Homing leucocytes et des migrations 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.

Évaluer la réponse stomatique à Live cellules bactériennes en utilisant l&#39;imagerie feuilles entières

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.

Utiliser l&#39;imagerie Bioluminescent chargé d&#39;enquêter sur une synergie entre Streptococcus pneumoniae Et virus Influenza A de souriceaux

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.

Phage »Bioluminescent&quot; Reporter pour la détection des bactéries pathogènes de la catégorie A

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.

La salive, les glandes salivaires, et Collection hémolymphe de tiques Ixodes scapularis

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 méthode d&#39;imagerie à distinguer inflammation aiguë et chronique

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.

Génération et multi-phénotypique criblage à haut contenu de<em&gt; Coxiella burnetii</em&gt; Mutants de transposon

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 et in vivo modèle pour étudier l&#39;adhésion bactérienne au navire muraux dans des conditions de débit

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.

Non invasif<em&gt; In Vivo</em&gt; Imagerie optique à fluorescence d&#39;une activité MMP inflammatoire à l&#39;aide d&#39;un agent d&#39;imagerie fluorescente activable