Ce travail a été soutenu par l&#39;Université du Wisconsin-Madison Herman I. Shapiro Distinguished Graduate Fellowship (CMT), l&#39;attribution des NIH AI072817 (AMP) et l&#39;American Heart Association Award 0840059N (LJK).

1. Préparation de fibroblastes de prépuce humain (HFF) enduit de lamelles <ol><li> Placer une lamelle de verre stérile ronde sur le fond des puits d&#39;une plaque de 24 puits de culture de tissu. </li><li> Pour récolter HFFS d&#39;une confluence 150cm 2 flacon, rincer le ballon deux fois avec du PBS 1X et ajouter 2,5 ml de 0,025% de trypsine-EDTA. Incuber flacon à 37 ° C pendant 5-10 minutes. </li><li> Exploiter le côté du ballon de contre la paume de votre main peut aider à détacher les cellules de la fiole. Une fois que les cellules ont libéré de la fiole, ajouter 150 ml de HFF moyennes (Dulbecco Modified Eagle Medium [DMEM] avec 10% de FBS, 2 mM de L-glutamine et 1% de pénicilline-streptomycine) et utiliser le moyen de se rincer la fiole et de recueillir les cellules. </li><li> Distribuer 1 ml de cellules par puits avec les lamelles. </li><li> Laisser les cellules deviennent confluentes à 37 ° C, 5% de CO 2. </li></ol> 2. Préparation L929 milieu conditionné (CM) pour le développement de BMC <ol><li> L929 est une ligne aneuploïdes murin fibrosarcome cellule qui sécrète colonies de macrophages facteur stimulant, après de longues périodes de confluence. Cette sécrétion permet à la CM de cellules L929 à être utilisé pour développer des cellules de la moelle osseuse de souris en macrophages dans une culture cellulaire. </li><li> Une fois que des cellules L929 sont confluentes, laissez-les incuber pendant une 7-9 jours supplémentaires jusqu&#39;à ce qu&#39;ils apparaissent sphériques et commencer à lever hors du ballon. Recueillir surnageant et le culot des cellules détachées à 425 xg pendant 10 minutes. Ce surnageant est la CM. </li><li> Un flacon de 150 cm 2 de cellules L929 rendements de 30 ml de CM, qui génère 150 ml de milieu de BMC. BMC milieu est composé de 10% de FBS, CM 20%, 1% de pénicilline-streptomycine et 1% de L-glutamine dans le DMEM. </li></ol> 3. L&#39;isolement de la moelle osseuse et de culture cellulaire de BMC <ol><li> Sacrifice C57BL / 6 souris en CO 2 et la surface de l&#39;asphyxie stériliser à 70% d&#39;éthanol. </li><li> Exposer le péritoine en soulevant la peau de l&#39;abdomen près de la jambe et la coupe avec des ciseaux. Évitez de perforer le péritoine. Couper à la jambe pour exposer le tibia et le fémur. Coupez le tissu musculaire de l&#39;os de la jambe à l&#39;aide des ciseaux. </li><li> Pour supprimer le fémur, couper une fois en dessous du genou et une fois près de la hanche. Placez les os dans un plat de Pétri contenant bactériologiques froide PBS 1X. Ne pas utiliser la culture de tissus traités boîtes de Pétri pour ces étapes parce que le BMC se conformer de façon permanente. Utiliser un scalpel pour gratter le tissu musculaire supplémentaire. Couper l&#39;os juste au-dessus du genou pour exposer la moelle. </li><li> Répétez les étapes (b) et (c) de recueillir la moelle osseuse du fémur secondes. </li><li> Utiliser une seringue de 10cc rempli de PBS 1X froid et d&#39;une aiguille de calibre 25 pour déloger la moelle osseuse dans un tube conique de 50 ml. L&#39;os devrait apparaître pur moelle blanche fois est retiré. </li><li> Passez le mélange PBS / la moelle par une aiguille de calibre 22 pour briser les agrégats de moelle osseuse. </li><li> Spin le mélange à 425 xg pendant 10 minutes, et retirer le surnageant. </li><li> Reprendre le culot dans 8 ml de milieu de BMC. Ajouter 1 ml de suspension cellulaire et 9 ml BMC moyenne à 8 bactériologiques boîtes de Pétri. </li><li> Incuber à 37 ° C, 5% de CO 2. </li><li> Au jour 5, ajouter 10 ml BMC moyenne à chaque assiette. Au jour 7, les cellules seront pleinement développées. BMC peuvent être repiquées pendant 1-2 semaines après la maturation. </li><li> Pour diviser BMC, éliminer le milieu et ajouter 5 ml de PBS 1X froid à chaque boîte de Pétri. Incuber à 4 ° C pendant 30 minutes, jusqu&#39;à ce que les cellules commencent à se soulever. Rincez le BMC de la plaque avec une pipette de transfert stérile. </li><li> Piscine du BMC dans un tube conique de 50 ml et de granulés à 425 xg pendant 10 minutes. </li><li> Reprendre le culot dans 10 ml de BMC moyen et compter sur un hématimètre. Graine 2x105 BMC par puits dans une plaque de 24 puits avec des lamelles de verre ronde sur le fond. Laissez les BMC adhèrent nuit avant d&#39;infecter avec T. gondii. BMC en excès peuvent être ensemencés sur des boîtes de Pétri pour une utilisation ultérieure. </li></ol> 4. Infecter les cellules avec T. gondii <ol><li> Infect 25 cm 2 flacon de HFFS confluence avec 2x10 6 T. gondii et grandir jusqu&#39;à ce que les cellules commencent à lyser (environ 2-3 jours). </li><li> Utilisez un grattoir pour enlever la cellule de l&#39;hôte infecté monocouche de cellules de la fiole de culture de tissus, puis relâchez les parasites de la cellule hôte par le passage de la monocouche délogé par une aiguille de calibre 27. </li><li> Utilisez un hématimètre pour déterminer le nombre de parasites dans le milieu. </li><li> Infect 10 5 parasites par bien avec lamelle de monocouches HFF de protocole 1, ou BMC du Protocole n ° 3. Laissez les parasites envahissent pendant 3 heures à 37 ° C, 5% de CO 2. </li></ol> 5. Initier T. gondii bradyzoite de développement par les contraintes environnementales <ol><li> Le développement de Bradyzoite HFFS avec une augmentation de pH du milieu et de CO 2 de famine <ol class="lalpha"><li> Préparer le milieu du développement. Développement à moyen contient RPMJ&#39;ai 1640 sans bicarbonate, 1% de FBS, 1% de pénicilline-streptomycine, et 42 mM d&#39;HEPES. pH à 8,0 et stériliser par filtration. </li><li> Retirer du DMEM HFFS infectés, rincer avec du PBS 1X, et ajoutez 1 ml de milieu de développement. </li><li> Incuber pendant 3 jours à 37 ° C avec l&#39;air ambiant. </li></ol></li><li> L&#39;activation du BMC <ol class="lalpha"><li> Préparer le milieu pour activer BMC. Moyennes activation est complétée par BMC moyennes d&#39;IFN-γ 100U/ml et 100 ng / ml de LPS. Soniquer LPS dans un bain d&#39;eau pendant 2 minutes avant de l&#39;utiliser pour perturber les agrégats. </li><li> Retirer BMC moyenne des puits et la remplacer par 1 ml de milieu d&#39;activation. </li><li> Incuber à 37 ° C, 5% de CO 2 pendant 3 jours. </li></ol></li></ol> 6. Détection par immunofluorescence <ol><li> Rincez infectés puits trois fois avec du PBS 1X. </li><li> Fixer le monocouche avec 200ul de formaldéhyde à 3% pendant 20 minutes. </li><li> Retirer fixateur et rincer les puits avec du PBS 1X. </li><li> Ajouter 200 ul de 0,1 M de glycine aux puits et laisser reposer pendant 5 minutes. </li><li> Retirez la glycine et rincer les puits avec du PBS 1X. </li><li> Ajouter 250 pi de 3% BSA/0.2% TritonX-100 dans du PBS 1X à perméabiliser et bloquer la monocouche pendant 30 minutes. </li><li> Retirer la solution de blocage et rincer les puits avec du PBS 1X. </li><li> Ajouter 50 ul d&#39;une dilution 1:250 de la rhodamine Dolichos biflorus agglutinine de 3% BSA/0.2% de Triton X-100 dans du PBS 1X. </li><li> Recouvrir la plaque et les placer sur la plate-forme agitateur à température ambiante pendant 1 heure </li><li> Lavez les lamelles 3 fois avec 0,2% de Triton X-100 dans du PBS 1X pendant 5 minutes chacun sur un shaker plate-forme. </li><li> Lamelles mont en utilisant le milieu de montage Vectashield contenant 4&#39;6-diamidino-2-phénylindole (DAPI). </li><li> Vitrail T. gondii peuvent être visualisées en utilisant un microscope à fluorescence avec un filtre approprié pour la rhodamine à un grossissement de 100x. </li></ol> 7. Les résultats représentatifs La figure 1 montre coloration DBA représentant de T. gondii dans BMMs activé et HFFS sous stress pH. Les deux taches DBA montrer autour du parasite contenant des vacuoles, indiquant la présence de composants de la paroi du kyste. L&#39;image montre BMM activé coloration DBA qui est conforme à la surface de la vacuole. La section transversale du T. gondii dans pH souligné HFFS montre la paroi du kyste sans structures internes tachés. <img src="/files/ftp_upload/2091/2091fig1.jpg" alt="Figure 1" /> Figure 1. Coloration de la DBA a souligné T. gondii. parasites intracellulaires dans des conditions de stress, BMMs activé ou pH souligné HFFS, ont été colorés avec DBA conjugué rhodamine (rouge). Contraste d&#39;interférence différentiel (DIC) montre le contour du kyste et la barre d&#39;échelle est égal à 2 uM noire. Expériences sur animaux Expériences sur les animaux ont été effectuées en conformité avec les directives et règlements établis par l&#39;Université du Wisconsin et de protection des animaux Comité utilisation.

<ol>
	<li>Tomida, M., Yamamoto-Yamaguchi, Y., Hozumi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+a+factor+inducing+differentiation+of+mouse+myeloid+leukemic+M1+cells+from+conditioned+medium+of+mouse+fibroblast+L929+cells.">Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells.</a> J Biol Chem. 259 (17), 10978-10980 (1984).</li><li>Matrajt, M., Donald, R. G., Singh, U., Roos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+differentiation+mutants+in+the+protozoan+parasite+Toxoplasma+gondii.">Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii.</a> Mol Microbiol. 44, 735-747 (2002).</li><li>Singh, U., Brewer, J. L., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tachyzoite+to+bradyzoite+differentiation+mutants+in+Toxoplasma+gondii+reveals+a+hierarchy+of+gene+induction.">Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction.</a> Mol Microbiol. 44, 721-733 (2002).</li><li>J, P., Knoll, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Toxoplasma+gondii+development+mutants+identifies+a+potential+proteophosphogylcan+that+enhances+cyst+wall+formation.+Molecular+and+Biochemical+Parasitology.">Isolation of Toxoplasma gondii development mutants identifies a potential proteophosphogylcan that enhances cyst wall formation. Molecular and Biochemical Parasitology.</a> 169 (2), 120-123 (2010).</li><li>Bohne, W., Heesemann, J., Gross, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Bradyzoite-Specific+Toxoplasma+gondii+Antigens+in+Gamma+Interferon-Treated+Mouse+Macrophages.">Induction of Bradyzoite-Specific Toxoplasma gondii Antigens in Gamma Interferon-Treated Mouse Macrophages.</a> Infection and Immunity. 61 (3), 1141-1145 (1993).</li><li>Ibrahim, H. M., Bannai, H., Xuan, X., Nishikawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+Cyclophilin+18-Mediated+Production+of+Nitric+Oxide+Induces+Bradyzoite+Conversion+in+a+CCR5-Dependent+Manner.">Toxoplasma gondii Cyclophilin 18-Mediated Production of Nitric Oxide Induces Bradyzoite Conversion in a CCR5-Dependent Manner.</a> Infection and Immunity. 77 (9), 3686-3695 (2009).</li><li>Lyons, R. E., McLeod, R., Roberts, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+tachyzoite-bradyzoite+interconversion.">Toxoplasma gondii tachyzoite-bradyzoite interconversion.</a> Trends in Parasitology. 18 (5), 198-201 (2002).</li><li>Berghaus, L. J., Moore, J. N., Hurley, D. J., Vandenplas, M. L., Fortes, B. P., Wolfert, M. A., Boons, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+primary+murine+macrophage-lineage+cells+and+RAW+264.7+cells+to+ligands+of+Toll-like+receptors+2,+3,+and+4.">Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4.</a> Comp Immunol Microbiol Infect Dis. , (2009).</li><li>Mordue, D. G., Scott-Weathers, C. F., Tobin, C. M., Knoll, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+patatin-like+protein+protects+Toxoplasma+gondii+from+degradation+in+activated+macrophages.">A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages.</a> Mol Microbiol. 63 (2), 482-496 (2007).</li><li>Bohne, W., Heesemann, J., Gross, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+replication+of+Toxoplasma+gondii+is+necessary+for+induction+of+bradyzoite-specific+antigens:+a+possible+role+for+nitric+oxide+in+triggering+stage+conversion.">Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion.</a> Infection and Immunity. 62 (5), 1761-1777 (1994).</li></ol>

Bien que le mécanisme de développement bradyzoite n&#39;est pas entièrement comprise, des analyses génétiques moléculaires de T. conversion de l&#39;étape gondii dans la culture de tissus a conduit à la découverte des gènes qui sont impliqués dans la formation de kystes bradyzoite 2,3,4. Les analyses ont également conduit à l&#39;observation que certains marqueurs bradyzoite sont exprimés dans d&#39;autres conditions de stress prolongé, y compris la croissance dans les macroph...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960-051</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11150</td>
<td>heat inactivate </td>
</tr>
<tr>
<td>Rhodamine Dolichos biflorus agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>RL-1032</td>
<td>&nbsp;</td>
<tr>
<td>Formaldehyde (16%)</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>18814</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP381-5</td>
<td>&nbsp;</td>
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<td>HEPES</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP310-1</td>
<td>&nbsp;</td>
<tr>
<td>IFN&gamma;</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>315-05</td>
<td>Store in single use aliquots</td>
</tr>
<tr>
<td>L-glutamine (200mM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Lipopolysaccharide (LPS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4391-1MG</td>
<td>Store in single use aliquots</td>
</tr>
<tr>
<td>Microscope Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-80 12CIR-1</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
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<td>RPMI medium 1640 powder (with L-glutamine, without bicarbonate)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>31800-022</td>
<td>&nbsp;</td>
<tr>
<td>Triton-X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP151-500</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA (0.25%)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>VectaShield mounting media with DAPI</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
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toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions

Toxoplasma gondii est un parasite intracellulaire obligatoire qui peut envahir toute cellule nucléée des animaux à sang chaud. Lors de l&#39;infection, T. gondii diffuse comme une forme rapide répliquant appelé tachyzoïtes. Tachyzoïtes convertir en une forme à croissance lente enkystées appelé bradyzoite par un processus de signalisation qui ne sont pas bien caractérisés. Dans les animaux, les kystes bradyzoite sont trouvés dans le système nerveux central et les tissus musculaires et représentent la phase chronique de l&#39;infection. Conversion à bradyzoïtes peut être simulé en culture de tissus par le CO 2 la famine, l&#39;aide moyenne avec un pH élevé, ou l&#39;ajout d&#39;interféron gamma (IFN). Bradyzoïtes sont caractérisées par la présence d&#39;un kyste, à laquelle la lectine de Dolichos biflorus agglutinine (DBA) lie. DBA marqué par fluorescence est utilisée pour visualiser la paroi du kyste chez des parasites cultivés dans des fibroblastes de prépuce humain (HFFS) qui ont été exposés à faibles émissions de CO 2 et moyennes pH élevé. De même, les parasites qui résident dans les os murins dérivés de la moelle macrophages (BMMs) affichent une paroi du kyste détectable par DBA après la BMMs sont activés avec IFN et de lipopolysaccharides (LPS). Ce protocole va montrer comment induire une conversion de T. gondii à l&#39;aide d&#39;un support bradyzoïtes pH élevé de croissance avec le CO 2 à faible et l&#39;activation de BMMs. Les cellules hôtes seront cultivés sur des lamelles, infectés par tachyzoïtes et soit activé avec plus d&#39;IFNy et LPS (BMMs) ou exposé à un milieu de croissance à pH élevé (HFFS) pendant trois jours. À la fin des infections, des cellules hôtes seront fixées, perméabilisées, et bloqués. Parois des kystes seront visualisés en utilisant la rhodamine DBA avec la microscopie à fluorescence.

Watch this Scientific Journal Video about Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions at JoVE.com

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii se convertit en une forme un kyste, en réponse aux stress environnementaux, ce qui peut être imité dans les modèles de culture de tissus. Cette vidéo montre des techniques pour examiner la formation des parois du kyste en activant la moelle osseuse dérivées des macrophages ou des changements de pH moyen de croissance des cellules de fibroblastes.

Toxoplasma gondii Formation paroi du kyste dans la moelle osseuse Activé macrophages dérivés et Conditions Bradyzoite

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

toxoplasma-gondii-cyst-wall-formation-activated-bone-marrow-derived

Research

JoVE Journal

Immunology and Infection

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

16.1K vues.  University of Wisconsin. Toxoplasma gondii se convertit en une forme un kyste, en réponse aux stress environnementaux, ce qui peut être imité dans les modèles de culture de tissus. Cette vidéo montre des techniques pour examiner la formation des parois du kyste en activant la moelle osseuse dérivées des macrophages ou des changements de pH moyen de croissance des cellules de fibroblastes.

Vidéo: Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Obtention hautement purifiée Toxoplasma gondii Les oocystes par un gradient de chlorure de césium discontinu

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Recherche

Immunologie et infection

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

Un crible génétique pour isoler Toxoplasma gondii Mutants Egress de la cellule hôte

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Enquête de la polarisation des macrophages aide de moelle osseuse macrophages dérivés

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Manipulation génétique<em&gt; Δku80</em&gt; Les souches pour l&#39;analyse génomique fonctionnelle de<em&gt; Toxoplasma gondii</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Dérivées de moelle osseuse Macrophages production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolement et injection intraveineuse de moelle osseuse de souris dérivées monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

Forward Génétique écrans à l&#39;aide pour identifier les macrophages<em&gt; Toxoplasma gondii</em&gt; gènes importants pour la résistance à l&#39;IFN-γ-cellulaire dépendant immunité autonome

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Caractérisation métabolique des polarisée M1 et M2 macrophages dérivées de moelle osseuse Utilisation en temps réel Analyse extracellulaire Flux

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Détection d’Activation Inflammasome et mort cellulaire de Pyroptotic dans les Macrophages dérivés de la moelle osseuse Murine

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

Isolement et culture in vitro de macrophages dérivés de la moelle osseuse pour l’étude de la biologie du NO-Redox

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...