Les auteurs tiennent à remercier le Dr Jay Pepose pour aider l&#39;enseignement du modèle pour nous. Ce travail a été soutenu par des subventions des National Institutes of Health EY11885 (PMS), EY21247 (PMS) et une subvention sans restriction de Research to Prevent Blindness au Département d&#39;ophtalmologie.

Remarque protection des animaux: Les analgésiques sont pas utilisées dans ce modèle car ils sont anti-inflammatoires et donc invalider le modèle. 1. Préparation de virus <ol><li> Cultiver HSV-1 sur des cellules Vero (80% de confluence) en utilisant une dilution à partir de 0,01 multiplicité d&#39;infection (en fiole T-150, d&#39;environ 5 x 10 5 unités formant plaque) pendant 3-4 jours jusqu&#39;à ce que toutes les cellules ont une rafle et sont facilement délogés en frappant le ballon. </li><li> Retirez le support pour tubes à centrifuger et centrifuger stériles dans Sorvall Legend RT à 4 ° C et 3500 rpm pendant 15 min. Après avoir retiré le surnageant (voir point 1.3), remettre en suspension grand culot de chaque tube de 50 ml avec 5 ml du total des médias. Ultrasons pendant 1 min en rafales deux 30 sec. </li><li> Retirer le surnageant et mettre en tubes de 50 ml stériles Oak Ridge et centrifuger à 9000 tours par minute, pendant 1 h à 4 ° C dans un Beckman J2-21 centrifugeuse. Il s&#39;agit de produire une petite pastille virale. Jeter le surnageant. </li><li> Combinez une sonication de 1,2 esprith à granulés de 1,3 et soniquer à nouveau pendant 45 secondes. Centrifugeuse à Sorvall Legend RT à 1500 rpm pendant 5 min à 4 ° C. Enregistrez le surnageant. Ceci constitue le stock de virus qui est aliquoté et conservé à -70 ° C. </li><li> Titre viral sur cellules Vero. J&#39;ai l&#39;habitude de plaque de 10 0 à 10 -8 à dilution (chacune étant une dilution de 10 fois) 2x à partir de 2 séries de dilution distincts. Titrage du virus est effectuée sur des plaques 12 puits stériles. </li></ol> 2. L&#39;infection de la souris <ol><li> Injecter chaque souris avec 0,1 ml d&#39;anesthésique cocktail/20 g de poids corporel de la souris (kétamine, 60 mg / kg de xylazine + 5 mg / kg, ce qui est dilué dans du HBSS). Ce cocktail est utilisé parce qu&#39;il est l&#39;un des moyens les plus sûrs de l&#39;anesthésie lors de dosages appropriés sont utilisés et les effets de cette drogue sont de très courte durée. </li><li> Lorsque les souris sont anesthésiées rayer la cornée de l&#39;oeil droit avec une aiguille de calibre 30 à l&#39;aide d&#39;un microscope à dissection pour faire en sorte que la cornée n&#39;est pas perforé. </li><li> Chaque rece sourisIVED une injection intrapéritonéale de 1 ml de sérum humain mis en commun (Sigma, Temecula, en Californie, anti-HSV réactivité avec une dose efficace pour la neutralisation virale de 50% de 1:800) afin de protéger la cornée des dommages pendant la primo-infection. </li><li> Nombre des souris avec des clips d&#39;oreille à l&#39;oreille l&#39;un ou coup de poing. </li><li> Après toutes les souris ont été numérotées; aide d&#39;une pipette 20 ul, nous infecter chaque œil avec 10 6 PFU (5 pi). Nous infecter un seul œil et l&#39;œil droit en général. Nous mettons également 5 ul de HBSS à l&#39;autre oeil pour le garder humide pendant qu&#39;ils sont anesthésiés. 5 ul est suffisant pour maintenir l&#39;hydratation des yeux, car les procédures sont exécutées en moins de 10 min, et les animaux sont dosés avec un anesthésique en conséquence. L&#39;infection est confirmée par écouvillonnage souris 3 jours post-infection avec un coton stérile qui a été hydraté dans 1 ml de milieu de Vero. Puis 100 ul est ajouté aux cellules Vero cultivées en plaques 48 puits. Ces plaques sont la surveillance quotidienne des effets cytopathiques<html> (Cpe). </li></ol> 3. La réactivation de la latence de souris <ol><li> Les souris sont anesthésiées au moins 5 semaines après la primo-infection. Il convient de noter que nous avons réactivé la souris jusqu&#39;à un an suivant la primo-infection et n&#39;ont pas observé de différences significatives dans la réponse. </li><li> Une fois anesthésié, ils sont placés TM20 Chromato-Vu transilluminateur, qui émet des UV-B à une longueur d&#39;onde maximale de 302 nm de telle sorte que seulement un seul oeil est exposé à la lumière UV-B. Chaque souris est exposé à la lumière 250 cm mJ d&#39;UV-B 2. </li><li> La réactivation est déterminée par écouvillonnage yeux avec applicateur de coton stérile qui est d&#39;abord placé dans 1 ml de milieu de Vero au jour 0 (avant l&#39;exposition UV-B pour tenir compte des excréteurs spontanées), puis sur les jours 1 à 7 post-irradiation UV-B. Le matériau tampon est évaluée comme décrit dans l&#39;2,5). Pour les prélèvements qui ont le virus infectieux de la quantité de virus est titré par une analyse de la plaque standard à l&#39;aide plaques de 12 puits. </li></ol>ove_title &quot;&gt; 4. Évaluation clinique <ol><li> Les souris sont évalués pour maladie de l&#39;œil clinique par un observateur masquée à l&#39;aide d&#39;un microscope binoculaire de dissection. </li><li> Opacité de la cornée est évalué sur une échelle de 0 à 4, où 0 indique clairement stroma, 1 indique une légère opacification stromale, 2 indique une opacité modérée avec des caractéristiques discernables iris, 3 indique une opacité dense, avec une perte de détails iris défini, sauf marges d&#39;élèves, et 4 indique opacité la plus totale, sans vue postérieure. </li><li> La néovascularisation est évalué sur une échelle de 1 à 8 en divisant la cornée en 4 quadrants égaux et déterminer l&#39;étendue de la vascularisation dans chacun de ces quadrants. </li><li> Blépharite est mesurée comme suit: 0, pas de lésions; 1, paupière gonflement minimal; 2, décharge modérée oculaire gonflement et croustillant; 3, un gonflement sévère, modérée perte de cheveux périoculaire, et des lésions cutanées, et 4, un gonflement sévère avec robinet yeux en croûte, sévère perte de cheveux périoculaire, et des lésions cutanées. </li><li> Preuve de l&#39;encéphalite included dos voûté, les cheveux ébouriffés, et des anomalies neurologiques évidentes. Les souris sont euthanasiées quand ils affichent des signes importants de l&#39;encéphalite. </li></ol> 5. Les résultats représentatifs Le modèle récurrent qui a été décrit ci-dessus a été publié par Shimeld, et al. 1-3. Ils ont testé et notre laboratoire a utilisé une version modifiée de ce modèle de publier de nombreux manuscrits sur les 4-15 ans. Le modèle a été utilisé pour définir le rôle que la cytokine interféron γ-jeux dans récurrente HSV-1 maladie 10. Comme le montre la figure 3, les souris dépourvues IFN affiché pire maladie de la cornée que ne souris de type sauvage 14. Il a également été utilisé avec succès pour définir la valeur thérapeutique du vaccin différent construit plusieurs 6,10,11. Notez que dans un cas, le vaccin testé a bien fonctionné prophylactique mais n&#39;a pas prouvé pour être thérapeutiquement efficace 6. Cependant,quand un vaccin incompétent pour la réplication virale a été testé, il était non seulement efficace en prophylaxie, mais il était aussi très efficace thérapeutiquement 10,11, voir figure 5 dans la référence 10. Plus récemment, nous avons utilisé ce modèle pour déterminer si les différences entre les sexes étaient détectables chez des souris subissant HSK récurrente. Comme la figure 1 le montre, des souris mâles C57BL / 6 afficher l&#39;opacité cornéenne beaucoup moins que ne le font C57BL / 6 femelles souris. De même, lorsque la néovascularisation cornéenne a été évaluée, les souris mâles ont démontré la croissance des vaisseaux beaucoup moins nouvelle dans la cornée que ne souris femelles (figure 2). Nous sommes en train de tester si ce phénotype est spécifique de la souche en effectuant une analyse similaire dans d&#39;autres souches de souris. Nous allons également déterminer si la différence est due à l&#39;absence de testostérone ou la présence d&#39;œstrogènes. <img alt="Figure 1" src="/files/ftp_upload/4276/4276fig1.jpg" /> Figure 1. Maladie de la cornée des souris C57BL mâle et femelle / 6 souches de souris à la suite d&#39;UV-B réactivation induite. Souris infectées de façon latente ont été amenés à réactiver avec UV-B et les souris ont été suivis pendant une opacité cornéenne pendant 35 jours. Le nombre de souris utilisées pour ces études étaient les suivants: hommes, n = 15; femmes, n = 15. Les résultats indiquent la moyenne ± SEM. * Il était significativement plus élevée induite par le virus maladie chez les souris C57BL / 6 femelles pour les jours de 14 à 35 (P = 0,001 à 0,01). <img alt="Figure 2" src="/files/ftp_upload/4276/4276fig2.jpg" /> Figure 2. Maladie de la cornée des souris C57BL mâle et femelle / 6 souches de souris à la suite d&#39;UV-B réactivation induite. Souris infectées de façon latente ont été amenés à réactiver avec UV-B et les souris ont été suivis pendant une néovascularisation cornéenne pendant 35 jours. Le nombre de souris utilisées pour ces études étaient les suivants: hommes, n= 15; femmes, n = 15. Les résultats indiquent la moyenne ± SEM. * Il était significativement plus élevée induite par le virus maladie chez les souris C57BL / 6 femelles pour les jours de 14 à 28 (P = 0,001 à 0,01).

<ol>
	<li>Shimeld, C., Hill, T. J., Blyth, B., Easty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+model+of+recurrent+herpetic+eye+disease+in+mice.">An improved model of recurrent herpetic eye disease in mice.</a> Curr. Eye Res. 8, 1193-1205 (1989).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+latent+infection+and+induction+of+recurrent+herpetic+eye+disease+in+mice.">Reactivation of latent infection and induction of recurrent herpetic eye disease in mice.</a> J. Gen. Virol. 71, 397-404 (1990).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+immunization+protects+the+mouse+eye+from+damage+after+herpes+simplex+virus+infection+by+limiting+spread+of+virus+in+the+nervous+system.">Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system.</a> J. Gen. Virol. 71, 681-687 (1990).</li><li>Laycock, K. A., Lee, S. F., Brady, R. H., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+murine+model+of+recurrent+herpes+simplex+viral+keratitis+induced+by+ultraviolet+B+radiation.">Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation.</a> Invest. Ophthalmol. Vis. Sci. 32, 2741-2746 (1991).</li><li>Keadle, T. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+ameliorates+corneal+disease+in+a+mouse+model+of+recurrent+herpetic+keratitis.">IL-10 ameliorates corneal disease in a mouse model of recurrent herpetic keratitis.</a> Microbial Path. 38, 13-21 (2005).</li><li>Keadle, T. L., Laycock, K. A., Miller, J. K., Hook, K. K., Fenoglio, E. D., Francotte, M., Slaoui, M., Stuart, P. M., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+recombinant+glycoprotein+D+subunit+vaccine+on+the+development+of+primary+and+recurrent+ocular+infection+with+herpes+simplex+virus+type+1+in+mice.">Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice.</a> J. Inf. Dis. 176, 331-338 (1997).</li><li>Keadle, T. L., Laycock, K. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinflammatory+cytokines+IL-1+and+TNF-a+are+required+for+recurrent+herpetic+keratitis+in+NIH+mice.">Proinflammatory cytokines IL-1 and TNF-a are required for recurrent herpetic keratitis in NIH mice.</a> Invest. Ophth. Vis. Sci. 41, 96-102 (2000).</li><li>Keadle, T. L., Usui, N., Laycock, K. A., Kumano, Y., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+cytokine+expression+in+a+murine+model+recurrent+herpetic+stromal+keratitis.">Corneal cytokine expression in a murine model recurrent herpetic stromal keratitis.</a> Ocular Immunol. Inflam. 9, 193-205 (2001).</li><li>Keadle, T. L., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++and+CD8++cells+are+key+participants+in+the+development+of+recurrent+herpetic+stromal+keratitis+in+mice.">CD4+ and CD8+ cells are key participants in the development of recurrent herpetic stromal keratitis in mice.</a> Microbial Path. 32, 255-262 (2002).</li><li>Keadle, T. L., Morrison, L. A., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+immunization+with+a+virion+host+shutoff-defective,+replication-incompetent+herpes+simplex+virus+type+1+strain+limits+recurrent+herpetic+ocular+infection.">Therapeutic immunization with a virion host shutoff-defective, replication-incompetent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection.</a> J. Virol. 76, 3615-3625 (2002).</li><li>Keadle, T. L., Laycock, K. A., Morris, J. L., Leib, D. A., Morrison, L. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+vaccination+with+vhs(-)+herpes+simplex+virus+reduces+the+severity+of+recurrent+herpetic+stromal+keratitis+in+mice.">Therapeutic vaccination with vhs(-) herpes simplex virus reduces the severity of recurrent herpetic stromal keratitis in mice.</a> J. Gen. Virol. 83, 2361-2365 (2002).</li><li>Keadle, T. L., Morris, J. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+aminoguanidine+on+primary+and+recurrent+ocular+herpes+simplex+virus+infection.">The effects of aminoguanidine on primary and recurrent ocular herpes simplex virus infection.</a> Nitric Oxide. 13, 247-253 (2005).</li><li>Stuart, P. M., Morris, J. E., Sidhu, M., Keadle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL3+protects+mice+from+corneal+pathology+during+recurrent+HSV-1+infection.">CCL3 protects mice from corneal pathology during recurrent HSV-1 infection.</a> Front. Biosci. 13, 4407-4415 (2008).</li><li>Keadle, T. L., Alexander, D. E., Leib, D. A., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+gamma+is+not+required+for+recurrent+herpetic+stromal+keratitis.">Interferon gamma is not required for recurrent herpetic stromal keratitis.</a> Virology. 380, 46-51 (2008).</li><li>Carr, D. J., Austin, B. A., Halford, W. P., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Interferon-gamma+by+an+adenovirus+vector+blocks+herpes+simplex+virus+Type+1+reactivation+in+vitro+and+in+vivo+independent+of+RNase+L+and+double-stranded+RNA-dependent+protein+kinase+pathways.">Delivery of Interferon-gamma by an adenovirus vector blocks herpes simplex virus Type 1 reactivation in vitro and in vivo independent of RNase L and double-stranded RNA-dependent protein kinase pathways.</a> J. Neuroimmunol. 206, 39-43 (2009).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Le modèle récurrent de kératite stromale herpétique (HSK) décrit ici permet à l&#39;enquêteur d&#39;étudier HSK humain dans un modèle qui est plus conforme à ce qui est observé au cours de la maladie humaine. Ainsi, les forces du modèle sont que la maladie se produit dans le cadre d&#39;un système immunitaire qui a déjà stimulé par la maladie pendant primaire. Étant donné que ces souris ont déjà une réponse immunitaire contre le HSV-1, les facteurs qui réactivent que la réponse immunitaire sont pr...

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Sigma</td>
			<td>S7023</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-C1</td>
		</tr>
		<tr>
			<td>Pen/ Strep</td>
			<td>Sigma</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290018</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td>Legend RT</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>52-21</td>
		</tr>
		<tr>
			<td>Transilluminatior</td>
			<td>UVP</td>
			<td>95-0452-02</td>
		</tr>
		<tr>
			<td>Sonicatior</td>
			<td>Branson</td>
			<td>Sonifier 450</td>
		</tr>
		<tr>
			<td>Oak Ridge Tubes</td>
			<td>Nalgene</td>
			<td>Z 720410</td>
		</tr>
		<tr>
			<td>Flasks- 7150</td>
			<td>Corning</td>
			<td>430823</td>
		</tr>
		<tr>
			<td>Plates- 12 well</td>
			<td>Corning</td>
			<td>CLS 3513</td>
		</tr>
		<tr>
			<td>Plates- 48 well</td>
			<td>Corning</td>
			<td>CLS 3548</td>
		</tr>
		<tr>
			<td>Sterile Conical Tubes- 30 ml</td>
			<td>Corning</td>
			<td>CLS 430828</td>
		</tr>
		<tr>
			<td>Sterile Cotton-tipped Applicators</td>
			<td>Fisher</td>
			<td>23-400-125</td>
		</tr>
		<tr>
			<td>30G Needles</td>
			<td>Becton-Dickinson</td>
			<td>305106</td>
		</tr>
		<tr>
			<td>25G Needles</td>
			<td>BD</td>
			<td>305122</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309604</td>
		</tr>
	</tbody>
</table>

recurrent herpetic stromal keratitis in mice, a model for studying human hsk

Maladie oculaire herpétique, appelée kératite herpétique stromal (HSK), est une infection potentiellement cécitante de la cornée qui se traduit par plus de 300.000 visites cliniques chaque année pour le traitement. Entre 1 et 2 pour cent de ces patients atteints de la maladie clinique constatez une perte de la vision de la cornée infectée. La grande majorité de ces cas sont le résultat d&#39;une réactivation d&#39;une infection latente par l&#39;herpès simplex virus de type I et non en raison d&#39;une maladie aiguë. Fait intéressant, l&#39;infection aiguë est le modèle le plus souvent utilisé pour étudier cette maladie. Toutefois, il a été estimé que le modèle récurrent de HSK serait plus représentatif de ce qui se passe au cours de la maladie clinique. Les modèles animaux récurrents pour HSK ont employé deux lapins et des souris. L&#39;avantage des lapins est qu&#39;ils éprouvent la réactivation de la latence absence de tout stimulus connu. Cela dit, il est difficile d&#39;étudier le rôle que de nombreux facteurs immunologiques jouer dans HSK récurrente parce que le modèle de lapin n&#39;a pas l&#39;immunitéressources méthodologiques et génétique de la souris a. Nous avons choisi d&#39;utiliser le modèle de souris pour HSK récurrente car elle a l&#39;avantage de l&#39;existence de nombreuses ressources disponibles et aussi savoir quand aura lieu la réactivation parce réactivation est induite par l&#39;exposition aux rayons UV-B. Jusqu&#39;à présent, ce modèle a permis à ces laboratoires de l&#39;utiliser pour définir plusieurs facteurs immunologiques qui sont importantes pour cette maladie. Il nous a également permis de tester à la fois thérapeutique et l&#39;efficacité du vaccin.

Watch this Scientific Journal Video about Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK at JoVE.com

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

La plupart des études de la maladie de la cornée herpétique utiliser un modèle d&#39;infection primaire. Cependant, la primo-infection par le HSV-1 habituellement, n&#39;aboutit pas à la maladie humaine. Nous décrivons ici un modèle récurrent de la maladie herpétique cornée, ce qui rapproche le plus de la maladie humaine.

Kératite herpétique récurrente stromale chez la souris, un modèle pour l&#39;étude de l&#39;homme HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

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Immunology and Infection

11.8K vues.  Saint Louis University. La plupart des études de la maladie de la cornée herpétique utiliser un modèle d'infection primaire. Cependant, la primo-infection par le HSV-1 habituellement, n'aboutit pas à la maladie humaine. Nous décrivons ici un modèle récurrent de la maladie herpétique cornée, ce qui rapproche le plus de la maladie humaine.

Vidéo: Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

La<em&gt; Citrobacter rodentium</emModèle de souris&gt;: Étudier Contributions des agents pathogènes et l&#39;hôte de la colite infectieuse

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Recherche

Immunologie et infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Une<em&gt; In vitro</em&gt; Modèle pour étudier l&#39;hétérogénéité de la différenciation des macrophages humains et de la polarisation

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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 ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolement de cellules murines ganglions lymphatiques stromales

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; Modèle pour l&#39;étude des interactions entre les droits du sang total et de l&#39;endothélium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Infection expérimentale avec<em&gt; Listeria monocytogenes</em&gt; En tant que modèle pour l&#39;étude de l&#39;hôte Réponses Interféron-y

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Méthodologies pour l&#39;étude<em&gt; B. subtilis</em&gt; biofilms comme un modèle pour Caractériser Inhibiteurs biofilm petites molécules

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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

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

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

Cytométrie en flux Analyse des cellules tueuses naturelles lytique Activité dans le sang humain

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

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analyse de 18FDG TEP/CT d’imagerie comme un outil pour étudier Mycobacterium tuberculosis Infection et traitement chez les Primates Non humains

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Implantation et suivi par PET/CT d'un modèle orthotopic du mésothéliome pleural humain chez les souris athymiques

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...