Ce travail a été soutenu par les Instituts canadiens de recherche en santé (MSH-95368, 89773 et MOP-MOP-106623 à CZ) et la Fondation canadienne pour l&#39;innovation. SMC est le récipiendaire d&#39;une Association des IRSC / canadienne de gastroentérologie de bourses postdoctorales. CZ est un chercheur des IRSC Nouveau.

1. Propagation Trichuris muris oeufs <ol><li> Pour générer de nouveaux lots d&#39;œufs de Trichuris muris, infecter 20 à 30 des souris immunodéficientes (ex-NOD.Cg Prkdc scid IL2RG tm1Wjl / SZJ (NSG) ou 129S6/SvEvTac-Rag2 tm1Fwa (RAG2 - / -)) ou des souris génétiquement sensibles ( par exemple. AKR / J) qui sont âgées de 6-8 semaines, avec environ 300 oeufs de Trichuris muris par gavage oral. </li><li> Après 32-35 jours le sacrifice de la souris par le CO 2 asphyxie. </li><li> Exposer la partie ventrale de la souris et humide de l&#39;abdomen avec de l&#39;éthanol à 70%. </li><li> En utilisant des pinces saisir la peau de l&#39;abdomen et de faire une petite incision à l&#39;aide des ciseaux. Coupez la peau pour exposer le contenu abdominal. </li><li> Identifier le caecum et l&#39;intestin proximal grande, et les supprimer. </li><li> Placez le caecum et le gros intestin proximal dans un plat de 10 cm de Pétri contenant Eagle modifié par Dulbecco (DMEM) avec 500 U / ml de pénicilline et 500 ug / ml de streptomycine (P / S). Couper le caecum ouverte pour exposer la lumière et les vers. En utilisant une pince cæcum lieu dans un bécher de tampon phosphate salin (PBS) et secouer délicatement pour enlever les excréments. </li><li> Retour au cæcum boîte de Pétri et en utilisant des pinces lisses incurvées (Roboz RS-5047) tirez délicatement vers et les placer dans un puits d&#39;une plaque de 6 puits contenant 5 ml de DMEM + P / S. </li><li> Répétez ce processus pour toutes les souris. </li><li> Mettez la plaque 6 puits dans un contenant Tupperware avec une serviette en papier humide pour maintenir l&#39;humidité et incuber à 37 ° C pendant 4 heures. </li><li> Après 4 heures supprimer un ver utilisant une pince incurvée lisse et les diviser en deux nouveaux puits de la plaque de 6 puits contenant 5 ml de DMEM + P / S. Récolte du contenu de la même origine et le placer dans un tube Falcon de 15 ml. Spin tubes à 3000 tpm et retirez et réservez surnageant comme antigène 4 h (peut être utilisé pour la restimulation des cellules après PBS dialyse, où les rendements en protéines typiques sont de 10-20 mg par 20 souris) et resuspendre œuf contenant le culot dans l&#39;eau du robinet en autoclave. </li><li> Mettez la plaque de 6 puits de retour à 37 ° C pendant la nuit. Retirez et jetez les vers. Récolte du contenu des puits dans un tube Falcon et tournent à 3000 rpm pendant 5 min. Retirer le surnageant et la réserve de l&#39;antigène pendant la nuit (peut être utilisé pour Trichuris Ag ELISA spécifiques d&#39;isotypes d&#39;anticorps après la dialyse PBS, où les rendements en protéines typiques sont de 10-20 mg par 20 souris) et contenant des oeufs Resuspendre le culot dans l&#39;eau du robinet en autoclave. </li><li> Combinez 4 heures et les oeufs une nuit et filtrer les œufs à travers le tamis 70μm de supprimer tout les vers restants, puis transférer dans un flacon de 75 cm2. Gardez ballon dans un endroit sombre et couvert de feuilles pendant 6 semaines à température ambiante (RT). </li><li> Lavez les oeufs dans de l&#39;eau du robinet à l&#39;autoclave et resuspendre à 50 œufs viables dans 50 pi. Pour ce faire, centrifugeuse oeufs à 3000 rpm pendant 5 min et remettre en suspension dans l&#39;eau du robinet ~ 50ml stérile. Retirer 50 oeufs ul et le placer sur une lame de verre. Utiliser le grossissement 40x compter le nombre de viabilité des oeufs embryonnés dans l&#39;ul 50. Diluer les œufs pour donner 50 œufs viables dans 50 pi. Pour les images d&#39;œufs viables s&#39;il vous plaît voir l&#39;étude de Kopper et Mansfield 16. </li></ol> 2. L&#39;infection aiguë des souris expérimentales avec Trichuris muris œufs (200 œufs / souris) <ol><li> Bien resuspendre oeufs et enlever un volume suffisant pour traiter le nombre de souris X 2 (par exemple pour 10 souris prendre 200 x 10 souris ul x 2 = 4 ml d&#39;oeufs) et les placer dans un tube de 14 ml pression bouchon. </li><li> Inverser le tube de 14 ml bouchon pression pour mélanger. Amener 200 pi d&#39;œufs dans une seringue ml sur une aiguille de taille appropriée d&#39;alimentation (pour les souris pesant 25 grammes, nous avons utilisé de calibre 18 1 1 / 2 &quot;d&#39;aiguilles). Dans un Scruff la main de la souris et en utilisant la main d&#39;autres administrent des œufs par gavage oral . </li><li> Attendre 21 jours. </li></ol> 3. De pêche expérimentale <ol><li> Le 21ème jour du sacrifice des souris en utilisant du CO 2. </li><li> Recueillir le sang de souris par ponction cardiaque, dans une seringue non hépariné, et conserver à 4 ° C. Le jour suivant, d&#39;isoler le sérum et le congeler à -20 ° C. Le sérum peut être utilisé pour la détection de Trichuris spécifiques IgG et IgE par ELISA. </li><li> Exposer la partie ventrale de la souris et humide de l&#39;abdomen avec de l&#39;éthanol à 70%. </li><li> En utilisant des pinces saisir la peau de l&#39;abdomen et de faire une petite incision à l&#39;aide des ciseaux. Coupez la peau et la membrane sous-jacente pour exposer le contenu abdominal. </li><li> Identifier l&#39;intestin grêle, le caecum et le colon. Prenez le caecum avec une pince et tirez hors de la cavité abdominale. Avec une autre paire de pinces poussez intestin grêle vers la droite exposer les ganglions mésentériques (mLNs). Retirez le mLNs en faisant attention de ne pas couper dans les intestins et les placer dans un faucon de 15 ml contenant 2 ml de médias. Si désiré, ces peuvent être traitées et restimulées in vitro (4x10 6 / ml dans 1 ml de plaques de 24 puits avec 1 ug / ml αCD3/CD28 ou avec 50μg/ml 4h Ag Trichuris) pendant 72 heures pour l&#39;analyse des cytokines par ELISA et f intracellulairecytométrie en bas. </li><li> Découpez le caecum et le colon. </li><li> En utilisant une pince, sortez pelotes fécales du côlon distal et le placer dans un tube de 2 ml Axygen. Congeler à -20 ° C. Pelotes fécales peuvent être analysés par western blot pour l&#39;expression de la résistine-like molecule-β (β-RELM). </li><li> Enlevez l&#39;extrémité du cæcum (~ 0,5 cm). Pour enlever les excréments maintenir la pointe du cæcum avec une pince et rincer avec du PBS en utilisant une seringue de 3 ml et de calibre 22 aiguille dans une boîte de Pétri. Placer dans du paraformaldéhyde 4% frais dans un tube de 5 ml Falcon bouchon clin d&#39;oeil. Conserver à 4 ° C. Elles peuvent être inclus en paraffine, coupés et montés sur des lames pour l&#39;analyse histologique. </li><li> Supprimer un morceau de côlon proximal (~ 1 cm) pour l&#39;ARN. Mettez l&#39;échantillon dans 2 ml Axygen tube avec 500 ul RNAlater. Conserver à 4 ° C. Ces échantillons peuvent être analysés pour l&#39;expression des ARNm par qPCR. </li><li> Placez le reste du cæcum dans une boîte de Pétri étiquetés et congeler à -20 ° C. </li></ol> 4. Énumération des Trichuris muris ver dans le caecum <ol><li> Retirer du congélateur et le caecum place dans une boîte de Pétri contenant de l&#39;eau ~ 5 ml. </li><li> Avec des ciseaux, couper le caecum ouverte pour exposer la lumière. </li><li> Tenez le caecum avec des pinces et agiter vigoureusement le caecum pour enlever la majorité des matières fécales. </li><li> Transfert cæcum à l&#39;autre boîte de Pétri contenant de l&#39;eau douce et en utilisant une pince incurvée gratter délicatement l&#39;IECS le tissu sous-jacent. </li><li> Transfert cæcum d&#39;un nouveau plat de Pétri contenant de l&#39;eau et vigoureusement grattez tissus restants avec une pince, déchirant le tissu en petits morceaux. </li><li> Utiliser un microscope à dissection pour compter tous les vers dans tous les 3 des boîtes de Pétri. Comme vous trouvez un ver retirez le plat pour assurer le même ver n&#39;est pas comptée deux fois. Si les vers sont endommagé (cassé dans la moitié) ne comptent que se termine fin ou épais pour obtenir un décompte précis. </li></ol> 5. Les résultats représentatifs Le modèle d&#39;infection par Trichuris muris permet aux chercheurs de quantifier la charge parasitaire et des réponses immunitaires systémiques, ainsi que d&#39;examiner histologiquement la réponse inflammatoire dans le cæcum distale. Lorsque la lumière du cæcum récolté est exposé, vers Trichuris peuvent être énumérés en utilisant un microscope à dissection (figure 1A-B). Les paramètres de l&#39;immunité à Trichuris également isotype d&#39;anticorps IgG1 commutation d&#39;(associé à l&#39;immunité de type Th2) chez les animaux résistants, et IgG2a (associé à l&#39;immunité de type Th1) chez les animaux sensibles (figure 1C). Expression de la coupe spécifique des cellules de protéines résistine-like molecule-β (β-RELM) est associée à la clairance de Trichuris et est détectable par Western blot (figure 2). La partie distale du caecum peut être coloré avec l&#39;acide périodique-Schiff (PAS) pour évaluer les réponses des cellules caliciformes et l&#39;étendue de l&#39;inflammation intestinale en réponse à l&#39;infection (figure 3). <img src="/files/ftp_upload/2774/2774fig1.jpg" alt="Figure 1"/> Figure 1. Quantification de l&#39;immunité à Trichuris muris. (A) Résistant (B6) des souris expulser muris Trichuris par jour 21 post-infection. En revanche, sensibles (AKR / J) les souris deviennent chroniquement infectés par Trichuris, résultant en l&#39;établissement du parasite. Chaque barre représente la moyenne ± SEM de 4-8 animaux par groupe. (B) vers sont comptés en utilisant un microscope à dissection. Panneau supérieur montre des vers dans l&#39;isolement, le panneau du bas montre les vers, en présence de l&#39;IECS et les fèces. (C) Trichuris spécifique des titres IgG2a sont déterminés par ELISA de sérum dilué (1:2 dilution, à partir de 01h20 et se terminant à la dilution 1:2560) sur des plaques recouvertes d&#39;antigène Trichuris excréteurs (O / N Ag, 5 ug / ml). Sensible AKR / J souris ont des niveaux considérablement plus élevés de Trichuris spécifiques IgG2a sérum que résistants souris B6. <img src="/files/ftp_upload/2774/2774fig2.jpg" alt="Figure 2"/> Figure 2. Apurement des Trichuris muris est associée à l&#39;expression de la coupe spécifique des cellules β des protéines RELM-7. RELM-β production peut être détectée à partir des pelotes fécales de Trichuris infectés résistants (B6), mais pas sensibles (AKR / J) chez la souris par une analyse Western blot. Chaque voie est représentatif d&#39;un seul animal, (N) = Naive, (Tm) = 21 jours Trichuris souris infectées. <img src="/files/ftp_upload/2774/2774fig3.jpg" alt="Figure 3"/> Figure 3. L&#39;examen histologique de l&#39;infection à Trichuris cæcum distale suivantes. Sections um 5-7 de la distale cæcum colorés avec le PAS. Hyperplasie des cellules caliciformes et de la production de mucus sont évidents dans résistants (B6), mais pas sensibles (AKR / J) des souris suite à une infection par Trichuris.

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Expériences sur les animaux ont été effectuées en conformité avec les directives et règlements établis par l&#39;Université de la Colombie-Britannique.

Ce protocole détaille une dose standard de haute aiguë Trichuris muris infection qui peut être modifiée au besoin par l&#39;investigateur. Par exemple, les souris peuvent être sacrifiés et les tissus récoltés à des jours différents. Afin de déterminer que les souris ont réussi à établir la charge parasitaire complète qu&#39;ils peuvent être sacrifiés au jour 14, date à laquelle toutes les souris doivent porter un fardeau de quelque 200 vers. Les souris peuvent également être infectés pendan...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Aiguilles alimentation animale (18 x 1 ½ &quot;) </td><td> Popper </td><td> 7912 </td><td></td></tr><tr><td> Pince courbée lisse </td><td> Roboz </td><td> RS-5047 </td><td></td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11965 </td><td></td></tr><tr><td> NOD.Cg-Prkdc scid IL2R gtm1Wjl / SZJ (NSG) </td><td> Jackson Laboratories </td><td> 005557 </td><td> Ce sont les souris, nous avons utilisé, cependant, aucune des souris immunodéficientes ou souche sensible devrait fonctionner. </td></tr><tr><td> RNAlater </td><td> Qiagen </td><td> 76104 </td><td></td></tr><tr><td> Tubes de 2 ml </td><td> Axygen </td><td> MCT-200-C </td><td></td></tr><tr><td> Tubes de 15 ml </td><td> Falcon </td><td> 352096 </td><td></td></tr><tr><td> 6 assiettes bien </td><td> Falcon </td><td> 353046 </td><td></td></tr><tr><td> Paraformaldéhyde </td><td> Sciences Electron Microscopy </td><td> 15710 </td><td></td></tr><tr><td> α-RELMβ d&#39;anticorps </td><td> PeproTech Inc </td><td> 0694270Rb </td><td></td></tr></tbody></table>

trichuris muris infection: a model of type 2 immunity and inflammation in the gut

Trichuris muris est un pathogène naturel des souris et est biologiquement et antigéniquement similaires aux espèces de Trichuris qui infectent les humains et le bétail 1. Œufs infectieux sont donnés par gavage oral, éclosent dans l&#39;intestin grêle distal, envahissent les cellules épithéliales intestinales (CEI) qui bordent les cryptes du cæcum et du côlon proximal et sur ​​la maturation des œufs des vers la libération dans l&#39;environnement 1. Ce modèle est un outil puissant pour examiner les facteurs qui contrôle l&#39;activation des cellules CD4 + T helper (Th), ainsi que des changements dans l&#39;épithélium intestinal. La réponse immunitaire qui se produit dans les souches résistantes consanguines, telles que C57BL / 6 et BALB / c, est caractérisée par des cytokines Th2 polarisées (IL-4, IL-5 et IL-13) et l&#39;expulsion des vers tout type Th1 associée de cytokines (IL -12, IL-18, IFN-γ) de promouvoir des infections chroniques chez AKR ​​génétiquement sensibles / souris J 2-6. Cytokines Th2 promouvoir des changements physiologiques dans le microenvironnement intestinale, y compris le roulement rapide des CEI, la différenciation des cellules caliciformes, le recrutement et l&#39;évolution de la perméabilité épithéliale et la contraction des muscles lisses, qui ont tous été impliqués dans l&#39;expulsion 7-15 ver. Ici, nous détaillons un protocole pour la propagation de Trichuris muris oeufs qui peuvent être utilisées dans des expériences ultérieures. Nous fournissons également une récolte expérimentale de l&#39;échantillon avec des suggestions pour l&#39;analyse post-infection. Globalement, ce protocole permettra aux chercheurs les outils de base pour réaliser un modèle d&#39;infection par Trichuris muris souris qui peut être utilisé pour traiter les questions relatives à la propension Th dans le tractus gastro-intestinal ainsi que des fonctions effectrices immunitaires des CEI.

Watch this Scientific Journal Video about Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut at JoVE.com

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection est un modèle de l&#39;immunité intestinale Th2 où les souris résistantes générer une réponse immunitaire protectrice Th2 et souris susceptibles de générer une réponse Th1 pathologique.

Trichuris muris Infection: Un modèle de type 2 Immunité et inflammation de l&#39;intestin

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

trichuris-muris-infection-model-type-2-immunity-inflammation

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Immunology and Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

16.2K vues.  University of British Columbia. Trichuris muris infection est un modèle de l'immunité intestinale Th2 où les souris résistantes générer une réponse immunitaire protectrice Th2 et souris susceptibles de générer une réponse Th1 pathologique.

Vidéo: Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsulaire sérotypage<em&gt; Streptococcus pneumoniae</em&gt; Par agglutination au latex

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

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Immunologie et infection

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

L&#39;isolement, la caractérisation et de l&#39;examen fonctionnel du Réseau de cellules gingivale immunitaire

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophiles Isolement et analyse pour déterminer leur rôle dans le lymphome cellulaire Sensibilité aux agents thérapeutiques

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Application Fluorescence Resonance Energy Transfer (FRET) pour examiner effecteur translocation efficacité par<em&gt; Coxiella burnetii</em&gt; Pendant siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Une méthode non-invasive et technique non-intensif pour Induction et phénotypage of Experimental pneumonie bactérienne chez les souris

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

coupe de précision de la souris Tranches Lung pour visualiser les cellules vivantes pulmonaire dendritiques

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Basées sur la fluorescence neuraminidase Essai d&#39;inhibition pour évaluer la sensibilité des virus de la grippe à la neuraminidase Inhibiteur Classe de Antiviraux

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Effets du goût signalisation suppression de protéine sur l’Inflammation intestinale dans un modèle de souris de la maladie inflammatoire de l’intestin

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Mise en place d’une Infection virale et analyse de l’Interaction hôte-Virus chez Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Le modèle d’Infection de teigne Galleria mellonella pour candidose disséminée

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...