Ce travail a été soutenu par numéros de subvention et de R01AI055001 R01AI082127 du NIAID à la GRN et le Prix SREB bourse de thèse à JLB.

1. Marqué à la fluorescéine dextrane et GSL I-isolectine B 4 injections veine faciale à l&#39;étiquette vascularisation embryonnaire <ol><li> Préparer FITC-dextran (50ug/mL) en tampon phosphate salin (PBS) ou GSL 1 - isolectine B 4 (20ug/200uL) dans du PBS dans un tube Eppendorf de 1,5 ml et chaud à 37 ° C. Ajouter 100 ul de stock 1,25 vert rapide / PBS à la solution de FITC-dextran (volume total 1 ml) et 180uL de stock 1,25 Fast Green / PBS à l&#39;une GSL - isolectine B 4 (200uL volume total), de sorte que la solution est visiblement bleu. </li><li> Disséquer E14.5 E18.5-embryons et le sac vitellin, en laissant le pédoncule allantoïdien (artère ombilicale et la veine) intacte. </li><li> Transfert des embryons à un nouveau plat Petrie (60 X 15 mm) et les plonger dans du PBS à température ambiante. </li><li> Position de l&#39;embryon de fournir une vue sagittale de la tête / le visage. Utiliser la micro pince à disséquer délicatement saisir l&#39;embryon, à la tête. </li><li> Utiliser une aiguille 30G, injecter 50ul FITC-dextran (50ug/mL) ou GSL 1 - isolectine B 4 (20ug de 200uL PBS) dans la veine faciale pointant l&#39;aiguille vers l&#39;arrière de la tête. </li><li> Lorsque le colorant est visible dans la veine ombilicale, retirez l&#39;aiguille et séparer l&#39;embryon de la tige allantoïdien (artère ombilicale et la veine). </li><li> Après injection, permettent de rester dans l&#39;embryon du PBS à température ambiante pendant 2-3 minutes afin que le colorant circule dans tout l&#39;embryon. </li></ol> 2. Whole-Mont d&#39;analyse de la vascularisation cutanée <ol><li> Après avoir laissé le colorant de circuler dans tout l&#39;embryon, prélever des échantillons de peau provenant de régions des membres, le dos et l&#39;estomac, etc 8,9. </li><li> Laver la peau de l&#39;échantillon dans du PBS froid, et de fixer à 4% PFA / PBS pendant 2 heures 8,9. Lavez 3 fois pendant 10 minutes dans chacune des 4 ml flacon transparent avec 2 ml de PBS froid. </li><li> Échantillon de peau Placer sur un microscope de diapositives et ajouter 100 l de montage de médias à chaque diapositive et un couvercle en verre. </li><li> Autoriser diapositives à sécher dans un espace de rangement sombre. </li><li> Passez à l&#39;étape la section «Acquisition d&#39;image» 2. </li></ol> 3. Multi-couleur étiquetage du thymus et de la vascularisation du cœur et des cellules périvasculaires cryocoupes (Continuer à partir de la section 1, l&#39;étape 7) <ol><li> Embryon de toute «Flash geler» dans l&#39;azote liquide. Les embryons peuvent être conservés et à -80 ° C jusqu&#39;à l&#39;analyse. </li><li> Alternativement, disséquer le thymus, les rincer dans du PBS 4 ° C, et fixer dans 2 ml de paraformaldéhyde à 4% (PFA) / PBS pendant 2 heures. Lavez 3 fois pendant 10 minutes dans du PBS froid, placez thymidine dans les PTOM, et de geler et de stocker jusqu&#39;à utilisation à -80 ° C. </li><li> Pour cryosectioning, répartis dans les PTOM sur «bloquer» une section et monter l&#39;embryon ou les organes disséqués / tissus pour la coupe. </li><li> Couper les tissus congelés en 10 um d&#39;épaisseur et de recueillir des sections sur des lames. </li><li> Fixer des sections dans l&#39;acétone pendant 5-10 minutes. Lavez 3 fois dans le froid du SCT. </li><li> Bloc de 10% de sérum d&#39;âne / SCT dans une chambre humide à température ambiante. </li><li> Incuber pendant 1 heure sections-nuit avec 100 pi d&#39;anticorps primaires dans une chambre humide à 4 ° C: dans cet exemple, nous utilisons rat anti-souris CD31 (1:100) à l&#39;endothélium étiquette, et de chèvre anti-souris PDGFR-β (1:100) pour marquer les cellules périvasculaires. Il est utile pour couvrir les diapositives avec des bandes de Parafilm couper individuellement afin de s&#39;assurer que l&#39;anticorps est réparti uniformément sur toute la section. </li><li> Après incubation avec l&#39;anticorps primaire, se laver les sections 3 fois dans le froid du SCT. Incuber avec 100 pi d&#39;anticorps appropriés secondaire pendant 30 minutes minimum. </li><li> Lavez 3 fois dans le froid du SCT. Ajouter 100 ul de montage des médias à chaque diapositive et un couvercle en verre. </li><li> Autoriser diapositives à sécher dans un espace de rangement sombre. </li><li> Passez à la section «Acquisition d&#39;image». </li></ol> 4. Multi-couleur étiquetage du thymus et de la vascularisation des cellules périvasculaires pour les sections vibratome (Continuer à partir de la section 1, l&#39;étape 7) <ol><li> Disséquer les lobes du thymus de l&#39;embryon et les rincer dans du PBS froid. </li><li> Fix thymus dans 4% PFA / PBS à température ambiante pendant 2 heures. </li><li> Laver dans du PBS-Triton X (0,15%) 3 fois, 10 minutes et thymidine place dans une cartouche en plastique petit et plongez dans 4% à faible fondre agarose / PBS (~ 4 ° C). Le thymus doit être en contact avec le fond de la cartouche. </li><li> Autoriser agarose à solidifier sur la glace (3-5 minutes). Utilisez une lame de rasoir pour couper l&#39;excès d&#39;agarose. Ajoutez de la colle sur le bloc vibratome et adhérer au bloc de l&#39;échantillon. </li><li> Ajouter PBS froid à bain d&#39;eau jusqu&#39;à ce vibratome l&#39;échantillon et la lame sont immergés. </li><li> Réglez la vitesse et l&#39;amplitude (amplitude élevée et à faible vitesse modérée est idéal pour les mous sections du thymus). L&#39;amplitude doit être réduite si les articles rupture due à une agitation excessive. </li><li> Coupez 50 sections um. </li><li> Utiliser un pinceau, de recueillir des sections dans une microplaque de 24 puits dans du PBS froid. </li><li> Bloc sections dans 500 pl de sérum d&#39;âne de 10% dans du PBS-Triton X (0,15%) pendant 30 minutes. </li><li> Sections Incuber pendant 8 heures à une nuit avec l&#39;anticorps primaire, comme les anti-CD31 et anti-PDGFR-β, Dans un endroit couvert de 24 puits de microplaques. </li><li> Lavez 3 fois en PBS-Triton X (0,15%) sur un total de 8 heures à 4 ° C. </li><li> Sections de bloc dans le sérum d&#39;âne de 10% dans du PBS-Triton X (0,15%) pendant 30 minutes. </li><li> Incuber sections pendant 8 heures pour une nuit à 4 ° C avec les anticorps appropriés secondaire. </li><li> Lavez 3 fois en PBS-Triton X (0,15%) sur un total de 8 heures à 4 ° C. </li><li> Re-fixer les échantillons dans 4% des PFA / PBS pendant 30 minutes sur la glace. </li><li> Lavez 3 fois en PBS-Triton X (0,15%) de plus de 30 minutes sur la glace. </li><li> Déshydrater les échantillons à travers un gradué MeOH / PBS-Triton série X: 25% de MeOH, MeOH 50%, 75% MeOH, et 100% de MeOH à 10 minutes pour chaque étape. Remplacer MeOH 100% avec MeOH frais après 10 minutes et incuber pendant 1 heure à température ambiante. </li><li> Dans un récipient en verre, mélanger Babb (Benzyl Alcool: Benzyl Benzoate) dans un rapport 1:2. Combinez BABB avec MeOH pour une concentration finale de 50% et BABB MeOH 50%. Incuber l&#39;échantillon à Babb: MeOH pendant 10-15 minutes. </li><li> Transférer l&#39;échantillon dans un récipient en verre à 100% et BABB incuber pendant 10-15 minutes ou jusqu&#39;à ce que dégagé, à température ambiante. </li><li> Remplissez glisser la dépression (0,7 mm de profondeur) avec BABB 100% frais et déguster le transfert vers le côté. Ajouter couvercle en verre (n ° 1.5) et sceller avec 2-3 couches de vernis à ongles. Laisser durcir le vernis à ongles dans le noir à température ambiante, puis stocker l&#39;échantillon à 4 ° C. Remarque: Les diapositives doivent être complètement scellé avant l&#39;acquisition de l&#39;image confocale. Les images doivent être acquises dans les 12-24 heures, comme des colorants fluorescents peuvent s&#39;estomper à Babb. </li><li> Passez à la section «Acquisition d&#39;image». </li></ol> 5. L&#39;acquisition des images <ol><li> Image 10 um coupes congelées avec un microscope confocal à l&#39;aide du Plan-Apochromat 20X/0.8 objectif (512 x 512 pixels) avec 488 - (FITC-dextran/GSL 1 - isolectine B 4), 543 -, et des lignes laser 633-nm. </li><li> Acquérir confocale z-sections de la peau monter l&#39;ensemble et 50 um d&#39;agarose-embedded sections en utilisant le Plan-Apochromat 10X/0.4 objectif (512 x 512 pixels) avec 488 - (FITC-dextran/GSL 1 - isolectine B 4), 543 -, et 633 nm lignes laser. Serial Z-sections doivent être prélevés de manière séquentielle au 1-micron pour chaque canal respectif. </li><li> Reconstruire série Z-sections à l&#39;aide Zeiss AxioVision 4.6 ou d&#39;autres logiciels d&#39;analyse d&#39;image. </li></ol> 6. Les résultats représentatifs: Marquage efficace de la vascularisation embryonnaire, est essentielle pour évaluer les défauts des vaisseaux sanguins chez des souris embryonnaire. La figure 1 montre un marquage spécifique des vaisseaux sanguins E16.5 thymus (1A-B) et co-marquage avec CD31 (1B), en plus de la coloration des ventricules droit et gauche (1E-F), respectivement. Le GSL I-B 4 isolectine protocole pour cryocoupes comme décrit dans les sections 1, 3 et 5 a été utilisée dans ces expériences. Whole-Mont étiquetage du réseau vaisseau sanguin cutané sur la souris E16.5, en utilisant les protocoles décrits dans les sections 1, 2 et 5 est représentée à la figure 1C-D. <img alt="Figure 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> Figure 1 Légende. FITC GSL I - isolectine B 4 injections veine faciale dans E16.5 embryons de souris. a. Cryosection du thymus embryonnaire suivant l&#39;injection. b. Fusion de CD31 co-marquage avec isolectine B 4. C. et D. entiers de montage de l&#39;injection de la peau vascularisation embryonnaire suivant. e. et f. Cryosection d&#39;embryon coeur e. (à droite ventricule) f. (ventricule gauche) après l&#39;injection.

<ol>
	<li>Cuddihy, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-mediated+cross-talk+within+the+neonatal+murine+thymus.">VEGF-mediated cross-talk within the neonatal murine thymus.</a> Blood. 113, 2723-2731 (2009).</li><li>Muller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+of+VEGF-A+in+thymus+epithelium+disrupts+thymus+blood+vessel+architecture.">Gene targeting of VEGF-A in thymus epithelium disrupts thymus blood vessel architecture.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 10587-10592 (2005).</li><li>Muller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+origin+of+perivascular+mesenchyme+in+the+adult+thymus.">Neural crest origin of perivascular mesenchyme in the adult thymus.</a> J. Immunol. 180, 5344-5351 (2008).</li><li>Foster, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+neural+crest-derived+cells+in+the+embryonic+and+adult+thymus.">Contribution of neural crest-derived cells in the embryonic and adult thymus.</a> J. Immunol. 180, 3183-3189 (2008).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+between+CCR7-+and+CCR9-mediated+chemokine+signals+in+prevascular+fetal+thymus+colonization.">Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization.</a> Blood. 108, 2531-2539 (2006).</li><li>Lavine, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+signals+regulate+a+wave+of+Hedgehog+activation+that+is+essential+for+coronary+vascular+development.">Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development.</a> Genes Dev. 20, 1651-1666 (2006).</li><li>Lavine, K. J., Kovacs, A., Ornitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+critical+for+maintenance+of+the+adult+coronary+vasculature+in+mice.">Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice.</a> J. Clin Invest. 118, 2404-2414 (2008).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Murphy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Notch4+signaling+induces+hallmarks+of+brain+arteriovenous+malformations+in+mice.">Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 10901-10906 (2008).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Whole-monter et PECAM-1 (CD31) sur la coloration des sections sont les méthodes classiques pour l&#39;étiquetage de la vascularisation de souris embryonnaires. Ces méthodes nécessitent l&#39;utilisation d&#39;immunofluorescence directe et / ou indirects, et des détergents pour perméabiliser les tissus de la souris. Cela s&#39;avère un processus plutôt rapide. Ici, nous avons employé FITC-dextran ou isolectine B 4 injections veine faciale pour marquer directement la vascularisation embryonnaire, élim...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> FITC-dextran </td><td> Sigma </td><td> FD150S-1G </td></tr><tr><td> Marqué à la fluorescéine GSL 1 - isolectine B 4 </td><td> Vector Laboratories </td><td> FL-1201 </td></tr><tr><td> Fast Green </td><td> MP Biomédical </td><td> 195178 </td></tr><tr><td> PFA </td><td> Fluka </td><td> 76240 </td></tr><tr><td> Sérum de veau fœtal </td><td> Atlanta Biologicals </td><td> S11550 </td></tr><tr><td> Optimal composé température de coupe (PTOM </td><td> VWR </td><td> 25608-930 </td></tr><tr><td> L&#39;acétone </td><td> JT Baker </td><td> 9006-33 </td></tr><tr><td> Sérum âne </td><td> Jackson </td><td> 017-000-121 </td></tr><tr><td> rat anti-souris CD31, </td><td> BD Pharmingen </td><td> 558736 </td></tr><tr><td> chèvre anti-souris PDGFR-β </td><td> R &amp; D Systems </td><td> AF1042 </td></tr><tr><td> d&#39;âne anti-CD31 de rat Alexa 647 (Invitrogen) </td><td> Biolegend </td><td> 102516 </td></tr><tr><td> d&#39;âne anti-chèvre Alexa 594 (Invitrogen) </td><td> Invitrogen </td><td> A11058 </td></tr><tr><td> Triton X -100 </td><td> Sigma-Aldrich </td><td> X-100 </td></tr><tr><td> Low Melt agarose / PBS </td><td> Sigma-Aldrich </td><td> A9414-25G </td></tr><tr><td> Méthanol </td><td> Fisher Scientific </td><td> A413-4 </td></tr><tr><td> Alcool benzylique </td><td> Acros scientifique </td><td> 148390010 </td></tr><tr><td> Benzoate de benzyle </td><td> Acros scientifique </td><td> 105860010 </td></tr><tr><td> Dépression diapositives </td><td> Fisher Scientific </td><td> S175201 </td></tr><tr><td> Fluorogel </td><td> Sciences Electron Microscopy </td><td> 17985-10 </td></tr><tr><td> Couverture en verre (22x22) -1,5 </td><td> Thermo Scientific </td><td> 152222 </td></tr><tr><td> Zeiss LSM 510 Meta microscope confocal </td><td> Zeiss </td><td></td></tr><tr><td> Micro pince à disséquer </td><td> Roboz </td><td> RS-5135 </td></tr><tr><td> Parafilm n ° OM992 </td><td> Fisher Scientific </td><td> 13-374-16 </td></tr><tr><td> 12 et 24 ainsi microplaques </td><td> Evergreen Scientific </td><td> 222-8044-01F </td></tr><tr><td> Lames de microscope Superfrost / Plus </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> 4mL flacons clairs </td><td> Scientifique national </td><td> B7800-2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

La mise en place d&#39;un réseau de vaisseaux sanguins fonctionnelle est une partie essentielle de l&#39;organogenèse, et est requis pour la fonction des organes optimale. Par exemple, dans la formation adéquate vascularisation du thymus et de structuration est indispensable pour l&#39;entrée dans l&#39;organe thymocytes matures et les cellules T de sortie vers la périphérie. La disposition spatiale des vaisseaux sanguins dans le thymus est dépendante des signaux du micro-environnement local, à savoir les cellules épithéliales thymiques (TEC). Plusieurs rapports récents suggèrent que la perturbation de ces résultats signaux dans la cuve 1,2 thymus défauts sang. Des études précédentes ont décrit les techniques utilisées pour étiqueter les 1,2 vascularisation néonatale et adulte du thymus. Nous démontrons ici une technique de marquage des vaisseaux sanguins dans le thymus embryonnaire. Cette méthode combine l&#39;utilisation de FITC-dextran ou Griffonia (Bandeiraea) lectine Simplicifolia I (GSL 1 - isolectine B 4) injections veine faciale et CD31 coloration des anticorps pour identifier les structures vasculaires du thymus et PDGFR-β à l&#39;étiquette du thymus périvasculaire mésenchyme 3-5. L&#39;option d&#39;utiliser cryocoupes ou sections vibratome est également fourni. Ce protocole peut être utilisé pour identifier les défauts du thymus vasculaire, ce qui est essentiel pour définir les rôles des molécules dérivées de TCE dans la formation des vaisseaux sanguins du thymus. Comme la méthode des étiquettes de la vascularisation entier, il peut aussi être utilisée pour analyser les réseaux vasculaires dans de multiples organes et de tissus dans l&#39;embryon, y compris la peau et le cœur 6-10.

Watch this Scientific Journal Video about A Method for Labeling Vasculature in Embryonic Mice at JoVE.com

A Method for Labeling Vasculature in Embryonic Mice

Cet article décrit une méthode pour l&#39;étiquetage de peau embryonnaire et les vaisseaux sanguins du thymus.

Une méthode pour l&#39;étiquetage dans les vaisseaux sanguins souris embryonnaires

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

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Immunology and Infection

15.5K vues.  University of Georgia. Cet article décrit une méthode pour l'étiquetage de peau embryonnaire et les vaisseaux sanguins du thymus.

Vidéo: A Method for Labeling Vasculature in Embryonic Mice

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 ...

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Immunologie et infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Inférence génotypique de tropisme du VIH-1 en utilisant la population basée séquençage du V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualisation virus de la dengue grâce à Alexa Fluor Étiquetage

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Non invasive d&#39;imagerie optique du système vasculaire lymphatique d&#39;une souris

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D imagerie multimodalité des<em&gt; Citrobacter rodentium</em&gt; Infections chez les souris

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

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 ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Induction de paralysie et de lésion du système visuel chez la souris par le particulier de cellules T pour Neuromyélite optique autoantigènes Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Une méthode pour la mesure de la fonction des glandes salivaires chez les souris

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Thrombose veineuse profonde induite par stase chez les souris surveillé par échographie de haute fréquence

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

Une méthode de nanoparticules argent pour améliorer le Syndrome de l’atrésie des voies biliaires chez les souris

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...