Nous souhaitons remercier Johnson &amp; Johnson, la TB Alliance, Astra Zeneca, Rib-X et Trius Therapeutics pour fournir bedaquiline, PA-824 (prétomanide), AZD5847, radezolid et tedizolid, respectivement. Brendan Prideaux, Matthew Zimmerman, Stephen Juzwin, Emma Rey-Jurado, Nancy Ruel, Leyan Li et Danielle Weiner ont soutenu l&#39;analyse MALDI, les méthodes bioanalytiques, la préparation du substitut de la casum, la synthèse chimique et l&#39;isolement de la casée de lapin. Ce travail a été mené avec le financement de la Fondation Bill et Melinda Gates, le prix # OPP1044966 et OPP1024050 à V. Dartois, Subvention d&#39;instrumentation partagée NIH S10OD018072, ainsi que le financement conjoint de la Fondation Bill et Melinda Gates et Wellcome Trust pour un Centre d&#39;excellence Pour l&#39;optimisation du plomb pour les maladies du monde en développement à P. Wyatt.

Toutes les études sur les animaux ont été effectuées conformément au Guide pour la prise en charge et l&#39;utilisation des animaux de laboratoire des Instituts nationaux de santé avec l&#39;approbation du Comité de soins et de soins pour animaux institutionnels du NIAID (NIH), Bethesda, MD. Toutes les études impliquant M. tuberculosis ont été effectuées dans un laboratoire avec un niveau de confinement de biosécurité 3 (BSL-3). 1. Modèle d&#39;infection de lapin et collection Caseum <ol><li> Infecte les lapins blancs de Nouvelle-Zélande avec M. tuberculosis en utilisant un système d&#39;exposition aux aérosols sans nez comme décrit précédemment. 12 , 13 Permettre à l&#39;infection de progresser pendant 12 à 16 semaines. Saisissez les lapins avec 35 mg / kg de kétamine et 5 mg / kg de xylazine par voie intramusculaire, euthanasiez les lapins avec 0,22 ml / kg de pentobarbital de sodium et de phénytoïne sodique par voie intraveineuse et poursuivez les nécropsies. </li><li> À l&#39;aide de pincettes et d&#39;un scalpel, retirez les poumons du coffre caVité. De chaque lobe pulmonaire, disséquer les cavités individuelles et les grands granulomes nécrotiques à l&#39;aide d&#39;un scalpel. Rassurer soigneusement le caséum des parois de la cavité et du granulome. Peser, enregistrer et stocker les échantillons dans des tubes à vis de 2 mL à -20 ° C jusqu&#39;à ce qu&#39;ils soient prêts à l&#39;emploi. </li><li> Rayons gamma des échantillons infectieux de caséum à 3 MegaRad sur de la glace carbonique pour les rendre inutilisables et sans danger pour l&#39;utilisation dans un laboratoire BSL-2. </li></ol> 2. Génération in vitro de la substitution de casée à partir de cellules THP-1 <ol><li> Cultiver des monocytes THP-1 dans du milieu RPMI 1640 (L-glutamine 2 mM et 10% de sérum bovin fœtal) dans des flacons de culture cellulaire T175 (80 ml / flacon). Incuber les flacons dans une atmosphère de CO2 à 5% à 37 ° C pendant 3 à 4 jours. </li><li> Centrifuger la culture d&#39;un flacon T175 dans deux tubes coniques de 50 ml à 150 xg pendant 5 min. Jeter le surnageant et suspendre la pastille dans 10 mL de média RPMI 1640. </li><li> Pipetter 5 μL de cette culture dans un tube de 1,5 mL contenant 45 μL de tRypan bleu. Bien mélanger en pipettant. Transférer 10 μL à un hémocytomètre et compter le nombre de monocytes THP-1 viables (non colorés) à l&#39;aide d&#39;un microscope optique (grossissement 10X). Calculer le nombre de cellules viables par ml de la culture. Diluez-le avec du milieu RPMI jusqu&#39;à la densité finale de 1,25 x 10 6 cellules / mL. </li><li> Chargez 40 mL de la culture sur une grande plaque de culture cellulaire (50 x 10 6 cellules / plaque). Ajouter 40 μL de PMA 100 μM (acétate de 12-myristate-13 de phorbol préparé dans l&#39;éthanol) et permettre aux cellules d&#39;adhérer pendant la nuit dans l&#39;incubateur. NOTE: La concentration finale de PMA est de 100 nM. </li><li> Diluer l&#39;acide oléique pur (OA) (0,89 g / mL) dans de l&#39;éthanol à la concentration de 0,1 M ( c&#39;est-à-dire 31,7 μL d&#39;OA dans 968,3 μl d&#39;éthanol). Diluer cette solution dans des milieux RMPI préchauffés frais à une concentration de 10 mM. Diluer cette suspension OA à 0,4 mM (concentration finale de travail) en milieu RPMI préchauffé à 37 ° C. </li><li> Supprimez les médias existants et non-adhered cellules provenant des plaques de culture de cellules et ajouter doucement 40 ml de 0,4 mM OA pour les macrophages THP-1 (THP-M). Incuber à 37 ° C dans l&#39;incubateur pendant une nuit. </li><li> Utilisez un microscope optique à un grossissement de 40x pour confirmer visuellement la présence de nombreuses inclusions de corps lipidiques dans chaque THP-M. Retirer tout le milieu RPMI à partir des plaques de culture de cellules et laver délicatement les cellules adhérentes deux fois avec une solution saline tamponnée au phosphate (PBS) en utilisant une pipette sérologique de 50 ml. REMARQUE: les corps Lipid apparaissent sous la forme de petites structures claires, sphériques dans le cytoplasme de la THP-M. </li><li> Ajouter 40 ml de 5 mM d&#39;acide éthylènediaminetétraacétique (EDTA) dans du PBS à chaque plaque. Incuber pendant 15 min à 37 ° C. </li><li> Détacher les macrophages spumeux (FM) en pipetant à plusieurs reprises de haut en bas sur la surface de toute la plaque en utilisant une pipette sérologique de 10 ml. Transférer la suspension cellulaire à un tube conique de 50 ml et centrifuger à 150 g pendant 5 min. </li><li> Remettre en suspension le culot cellulaire dans 10 ml de PBS (troisième lavage PBS) unEt transfert à un tube conique pré-pesé de 15 ml. Retournez à 150 xg pendant 5 min. Aspirer soigneusement le surnageant à l&#39;aide d&#39;une pipette sérologique et jeter. </li><li> Suivez les pastilles de FM à 3 cycles de congélation-décongélation pour lyser les cellules et les incuber à 75 ° C pendant 20-30 minutes pour dénaturer les protéines dans la matrice. Conservez les granulés à -20 ° C jusqu&#39;à ce qu&#39;ils soient prêts à l&#39;emploi. </li></ol> 3. Essai de dialyse à équilibrage rapide (ROUGE) <ol><li> Préparer des solutions stock 10 mM de tous les composés d&#39;essai dans le diméthylsulfoxyde (DMSO). Diluer à 500 μM de solutions de travail dans le DMSO avant chaque dosage. </li><li> Pesez le tube contenant la pastille de substitution Caseum. Soustraire le poids du tube vide pour obtenir le poids de la pastille seule. Ajouter 2-3 grains de métal par tube et, en utilisant un homogénéisateur de tissu à 1200 coups / min pendant 1 min, perturber le caséum ou la matrice de substitution dans le PBS (1: 9 w / v) pour obtenir une suspension 10x diluée de chaque matrice. </li><li> Spike 6,5 μL des 500uM solution du composé d&#39;essai dans 643,5 ul de l&#39;homogénat pour obtenir la concentration finale de 5 uM (≤1% de DMSO) et vortex. </li><li> Placez les inserts rouges dans la plaque de base. Ajouter 200 ul de la matrice de médicament-dopés dans la chambre de donneur (anneau rouge) de chaque insert RED et 350 ul de PBS dans chaque chambre de récepteur. Préparer 3 inserts pour chaque composé d&#39;essai (échantillons en triple). plaque d&#39;étanchéité avec un joint d&#39;étanchéité de plaque adhésif et incuber à 37 ° C sur le Thermomixer à 200 tours par minute (1 xg) pendant 4 h. </li><li> Après l&#39;incubation, mélanger doucement le contenu des chambres de donneur et receveur par pipetage vers le haut et vers le bas 2-3 fois. Pipeter 20 uL à des aliquotes d&#39;homogénat à partir des chambres de donneur et ajouter à 20 pl de PBS pur dans un tube de 1,5 ml (1: 1). De même, à partir des chambres de réception et ajouter à 20 pl d&#39;homogénat propre (adaptation de la matrice) sur la pipette des aliquotes de 20 pi d&#39;échantillons de PBS. 8 REMARQUE: la matrice de correspondance élimine le neced 2 courbes d&#39;étalonnage séparées (dans l&#39;homogénat et PBS) à effectuer une analyse quantitative. Contenu de la chambre de donneur peuvent sédimenter au cours du temps. Mélanger délicatement le contenu par pipetage avant de retirer aliquotes. </li></ol> 4. LC-MS Quantification et analyse des données <ol><li> Ajouter 160 ul de 1: 1 methanol: acétonitrile contenant 500 ng / mL de diclofénac ou 10 ng / mL verapamil (étalon interne) à chaque tube et vortexer pour précipiter les protéines. Centrifuger à 10 000 xg pendant 5 minutes pour sédimenter le précipité et on surnageants transfert en plaques de 96 puits à puits profonds pour l&#39;analyse de Chromatographie spectrométrie de masse liquide (LCMS). 7 </li><li> Construire des courbes d&#39;étalonnage à partir de 1-1,000 nM pour chaque composé d&#39;essai tout en maintenant la même composition de matrice que les échantillons ci-dessus. Quantifier la concentration du composé d&#39;essai dans les échantillons provenant des chambres de donneur et receveur au moyen d&#39;un procédé LC-MS. </li><li> Calculer la fraction non liée (f u) oF le médicament dans la matrice diluée en utilisant l&#39;équation 1. Calculer le f u dans la matrice non diluée en utilisant l&#39;équation 2 ( D = facteur de dilution de 10). 14 <img alt="L&#39;équation 1" src="/files/ftp_upload/55559/55559eq1.jpg" /> <img alt="L&#39;équation 2" src="/files/ftp_upload/55559/55559eq2.jpg" /></li><li> Vérifier la récupération (masse) de chaque composé en utilisant l&#39;équation 3 pour identifier les composés avec des problèmes de stabilité / métabolisme / liaison non spécifique. NOTE: Le recouvrement tombe généralement entre 70% et 130%. 15 <img alt="L&#39;équation 3" src="/files/ftp_upload/55559/55559eq3.jpg" /></li></ol>

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Il n&#39;y a pas d&#39;intérêts financiers concurrents.

Les lésions et les cavités nécrotiques pulmonaires chez les patients infectés par la tuberculose contiennent des sous-populations de bactéries qui sont récalcitrantes au traitement médicamenteux. Les carottes caséoises de ces structures sont particulièrement responsables d&#39;abriter ces persisters dans un environnement extracellulaire. 16 La répartition favorable des agents anti-bactériens dans ces endroits éloignés est considérée comme un déterminant important de l&#39;efficaci...

Le traitement de la maladie de la tuberculose pulmonaire exige une distribution efficace des médicaments dans différents types de lésions. des lésions nécrotiques et les cavités contiennent des centres caséeux qui abritent des sous-populations de bactéries tolérantes à la drogue ou « persistant ». 1, 2 La maladie cavitaire est associée à des taux de guérison inférieurs et un mauvais pronostic. 3, 4 études antérieures ont montré, en utilisant des techniques quantitatives et d&#39; imagerie, que la capacité de pénétrer caseum varie considérablement d&#39;une classe de médicaments à l&#39; autre. 5, 6 Ces méthodes nécessitent cependant l&#39;utilisation de modèles d&#39;infection des animaux qui sont lents et fastidieux. Un dosage in vitro qui mesure la liaison au médicament ex vivo caseum a été conçu. Cette liaison a été trouvé en corrélation inverse avec la pénétration des médicaments dans les granulomes caséeux et, par conséquent, estutilisé comme un outil de prédiction. 7 dialyse d&#39;équilibre est considéré comme l&#39;approche standard de l&#39;or à des études de liaison aux protéines plasmatiques. Le dispositif de dialyse d&#39;équilibre rapide (RED) offre un moyen rapide, facile à utiliser et système fiable pour effectuer ces tests. 8 Le dispositif est constitué de deux composants: à usage unique, jetable , comprenant des inserts 2 chambres séparées par un cylindre vertical de la membrane semi-perméable; et les plaques de base réutilisable qui peut contenir jusqu&#39;à 48 inserts à la fois. La membrane de dialyse a un qui est idéal pour des études de liaison à la drogue macromolécule de coupure 8 kDa de poids moléculaire (MWCO). Le rapport des volumes zone superficielle élevée du compartiment de membrane permet une dialyse rapide et équilibration. Les deux inserts et la plaque de base ont été validés pour une liaison non spécifique minimale. La combinaison du dispositif RED avec des techniques bioanalytiques fournit des estimations précises des fractions non liées de médicaments en pLasma. 8, 9 Bien que conçu à l&#39;origine pour mesurer liaison aux protéines plasmatiques, le dispositif RED a été utilisé dans plusieurs études de liaison de tissus en utilisant broyats. 10, 11 Dans ce protocole, on mesure la drogue se liant à caseum, les débris nécrotiques excisée des lésions nécrotiques et des cavités de lapins infectés par la tuberculose. Le acellulaire et la nature non-vasculaire caséeuse le rend facile à homogénéise en une suspension homogène qui est compatible avec le dosage. Étant donné que caseum est fastidieux à produire et difficiles à trouver, le protocole a été validé pour une utilisation avec une matrice de substitution qui est préparé à partir de macrophages spumeux. macrophages dérivés de monocytes THP-1 sont induits par l&#39;acide oléique pour accumuler plusieurs corps lipidiques qui leur donnent leur aspect « mousseuse ». Ces cellules chargées de lipides sont récoltées etTraité pour produire une matrice que nous utilisons comme substitut au caséum. Cette étude a montré que la liaison du médicament à cette matrice de substitution est bien corrélée à la liaison avec le caséum, imitant efficacement le processus in vivo qui entrave la pénétration du médicament dans le noyau caséo de granulomes et de cavités.

<table border="0" cellpadding="0" cellspacing="0" width="960">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">New Zealand White Rabbits</td>
			<td style="width:203px;">Covance</td>
			<td style="width:147px;">-</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">HN878 Mycobacterium tuberculosis</td>
			<td style="width:203px;">BEI Resources</td>
			<td style="width:147px;">NR-13647&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ketathesia (Ketamine) 100mg/mL C3N</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">56344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Anased (Xylazine) 100mg/mL</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">33198</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Euthasol (pentobarbital sodium and phenytoin sodium) Solution&#160;</td>
			<td style="width:203px;">Virbac</td>
			<td style="width:147px;">710101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">THP-1 monocytic cell line</td>
			<td style="width:203px;">ATCC</td>
			<td style="width:147px;">ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">175cm&#178; TC-Treated Flask (T175)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-3400-175</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 media w/o glutamine</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>MT-15-040-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone Fetal Bovine Serum, Gamma irradiated&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3091003IR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone L-glutamine, 200mM</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3003401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellstar TC dish, 145x20mm, vented</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-2881-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate13-acetate (PMA)&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>BP685-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:203px;">Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oleic acid&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>ICN15178101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Reusable Base Plate</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Inserts, 50/box&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED insert removal tool&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>89812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adhesive plate seal</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>08-408-240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH7.4, 10 X 500 mL (Gibco)&#160;</td>
			<td style="width:203px;">Life Technologies&#160;</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">DMSO</td>
			<td style="width:203px;">Sigma</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Acetonitrile</td>
			<td style="width:203px;">Sigma</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Methanol</td>
			<td style="width:203px;">Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Verapamil hydrochloride</td>
			<td style="width:203px;">Sigma</td>
			<td>V4629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Diclofenac sodium salt</td>
			<td style="width:203px;">Sigma</td>
			<td>93484</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-250-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ethanol, 200 proof</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">2010 Geno/Grinder</td>
			<td style="width:203px;">SPEX SamplePrep</td>
			<td style="width:147px;">2010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Bead Mill Homogenizer Accessory, Metal Bulk Beads</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-340-158</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">484R Cobalt 60 Irradiator</td>
			<td style="width:203px;">JL Shepard</td>
			<td style="width:147px;">7810-484-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">INCYTO&#160;C-Chip Disposable Hemacytometers</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:147px;">22-600-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Upright Light Microscope</td>
			<td style="width:203px;">Leica</td>
			<td style="width:147px;">DM1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Binary Liquid Chromatography system</td>
			<td style="width:203px;">Agilent</td>
			<td style="width:147px;">1260</td>
			<td>Multi-compenent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Mass spectrometer</td>
			<td style="width:203px;">AB Sciex</td>
			<td>4000</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro caseum binding assay that predicts drug penetration in tuberculosis lesions

L&#39;éradication de la maladie de la tuberculose nécessite des schémas thérapeutiques qui peuvent pénétrer dans les couches multiples de lésions pulmonaires complexes. La distribution des médicaments dans les noyaux caséeuses des cavités et des lésions est particulièrement crucial car ils abritent des bactéries sous-populations tolérantes médicaments communément appelés persisters. Les procédés existants pour la mesure de la pénétration du médicament dans les lésions de la tuberculose comportent in vivo coûteuses et longues études pharmacocinétiques couplés à des techniques de bioanalyse ou d&#39; imagerie. A été proposé La mesure in vitro du médicament se liant à des macromolécules caseum comme une alternative à ces techniques étant donné que cette liaison entrave la diffusion passive de molécules de médicament à travers caseum. dialyse d&#39;équilibre rapide est un système rapide et fiable pour effectuer des protéines plasmatiques et des études de liaison de tissus. Dans ce protocole, nous avons utilisé un dispositif de dialyse d&#39;équilibre rapide (RED) pour mesurer la liaison à la drogue broyats de caseum qui est EXCISDes lésions et des caries des lapins infectés par la tuberculose. Le protocole décrit également comment générer une matrice de substitution à partir de macrophages THP-1 chargés de lipides à utiliser à la place du caséum. Cet essai de liaison caseum / substitution est un outil important dans la découverte de médicaments contre la tuberculose et peut être adapté pour aider à étudier la distribution de médicaments dans des lésions ou des abcès causés par d&#39;autres maladies.

À l&#39;aide de ce protocole, nous avons testé des centaines de composés de développement de médicaments contre la tuberculose pour leur efficacité prédite à la casée pénétrante. La figure 1 permet de visualiser les concepts de base du dosage RED. La membrane de dialyse des inserts RED permet aux petites molécules non liées de se diffuser du puits du donneur au bien récepteur, atteignant finalement un équilibre entre les deux compartiments. Les petites mol...

Watch this Scientific Journal Video about An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions at JoVE.com

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Nous décrivons ici une méthode de dialyse à l&#39;équilibre rapide (RED) pour mesurer la liaison du médicament à la caséine à partir de lésions et cavités de la tuberculose pulmonaire. Le protocole est également utilisé avec une matrice dérivée de macrophages en mousse qui est un substitut efficace à la casée.

Un<em&gt; In Vitro</em&gt; Ensemble de liaison de caséine qui prédit la pénétration des drogues dans les lésions tuberculeuses

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

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Pharmacokinetics and Pharmacodynamics

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Unit 1

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Nonlinear Pharmacokinetics

Introduction to Respiratory System Drugs

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11.6K vues.  Rutgers.  Nous décrivons ici une méthode de dialyse à l'équilibre rapide (RED) pour mesurer la liaison du médicament à la caséine à partir de lésions et cavités de la tuberculose pulmonaire. Le protocole est également utilisé avec une matrice dérivée de macrophages en mousse qui est un substitut efficace à la casée. 

Vidéo: An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

Dans la formation de biofilms in vitro dans une diapositive Chambre de 8 puits

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Recherche

Immunologie et infection

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Drug faciliter la découverte: Un test automatisé inflammation Haute-contenu chez le poisson zèbre

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitative<em&gt; In vitro</em&gt; Test pour mesurer l&#39;adhésion des neutrophiles à Activé cellules endothéliales microvasculaires humaines primaires dans des conditions statiques

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modèle expérimental pour étudier la tuberculose-paludisme co-infection à transmission naturelle de Mycobacterium tuberculosis Et Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Étude des effets des probiotiques sur la colonisation pneumococcique l&#39;aide d&#39;un<em&gt; In Vitro</em&gt; Respect de dosage

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Faisant preuve d&#39;une multi-résistante<em&gt; Mycobacterium tuberculosis</em&gt; Amplification de puces à ADN

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Une méthode de référence de microdilution de bouillon pour dalbavancine<em&gt; In Vitro</em&gt; Tests de sensibilité des bactéries qui se développent Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Mise en place d&#39;un programme d&#39;installation à haut débit pour le criblage de petites molécules qui modulent c-di-GMP Signalisation dans<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Un haut-débit Assay Compatible pour évaluer l&#39;efficacité des médicaments contre Macrophage repiquées<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Méthodes in vitro pour la comparaison de liaison cible et CDC induction entre les anticorps thérapeutiques: Applications dans l&#39; analyse biosimilarité

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...