Este trabajo fue apoyado por la Universidad de Wisconsin-Madison Herman I. Shapiro distinguida Graduate Fellowship (CMT), premio NIH AI072817 (AMP) y la Asociación Americana del Corazón Premio 0840059N (LJK).

1. Preparación de fibroblastos de prepucio humano (HFF) recubierto cubreobjetos <ol><li> Coloque un cubreobjetos de vidrio estériles redonda sobre el fondo de los pozos de una placa de tejido de 24 pocillos. </li><li> Para la cosecha de un HFFS confluentes 150 cm 2 matraz, enjuagar el frasco dos veces con PBS 1X y añadir 2,5 ml de 0,025% de tripsina-EDTA. Incubar matraz a 37 ° C durante 5-10 minutos. </li><li> Tocando el lateral del recipiente contra la palma de su mano puede ayudar a separar las células del frasco. Una vez que las células se han dado de alta del matraz, añadir 150 ml de HFF medio (Dulbecco modificada de Eagle Medium [DMEM] con 10% de SFB, 2 mM L-glutamina y 1% de penicilina-estreptomicina) y usar el medio para enjuagar el frasco y recoger las células. </li><li> Dispensar 1 ml de células por pocillo con el cubreobjetos. </li><li> Permiten a las células para convertirse en confluentes a 37 ° C, 5% de CO 2. </li></ol> 2. Preparación L929 medio condicionado (CM) de BMC desarrollo <ol><li> L929 es un fibrosarcoma murino aneuploides línea de células que secreta el factor estimulante de colonias de macrófagos después de largos períodos de confluencia. Esto permite que la secreción de las células L929 CM a ser utilizado para desarrollar células de médula ósea de ratón se convierten en macrófagos en cultivo celular 1. </li><li> Una vez que las células L929 son confluentes, que los incuban por un período adicional de 7-9 días hasta que aparecen esférico y comenzar a levantar de la botella. Recoger el líquido sobrenadante y pellet cualquier célula independiente a 425 xg durante 10 minutos. Este sobrenadante es el CM. </li><li> Un frasco de 150 cm2 de células L929 rinde 30 ml de la CM, que genera 150 ml de medio de BMC. BMC medio está compuesto de 10% de SFB, 20% de CM, el 1% de penicilina-estreptomicina, y el 1% de L-glutamina en DMEM. </li></ol> 3. El aislamiento de la médula ósea y cultivo de células de BMC <ol><li> Sacrificio de un C57BL / 6 ratones por asfixia de CO 2 y la superficie de esterilizar con un 70% de etanol. </li><li> Exponer el peritoneo, levantando la piel del abdomen, cerca de la pierna y cortar con tijeras. Evitar perforar la cavidad peritoneal. Reducir a la pierna para exponer la tibia y el fémur. Cortar el tejido muscular de los huesos de las piernas con unas tijeras. </li><li> Para eliminar el fémur, una vez cortadas por debajo de la rodilla y una vez cerca de la cadera. Coloque los huesos en una placa de Petri que contiene bacteriológica frío PBS 1X. No utilice el cultivo de tejidos tratados con placas de Petri para estas medidas porque los PMP se adhiere de forma permanente. Use un bisturí para raspar el tejido muscular extra. Cortar el hueso justo por encima de la rodilla para exponer la médula ósea. </li><li> Repita los pasos (b) y (c) para recolectar la médula ósea del fémur segundos. </li><li> Use una jeringa de 10cc llena de frío PBS 1X y una aguja de calibre 25 para desalojar a la médula ósea en un tubo cónico de 50 ml. El hueso debe aparecer pura médula, una vez el blanco es eliminado. </li><li> Pase la mezcla de PBS / médula ósea a través de una aguja de calibre 22 para romper los agregados de médula ósea. </li><li> Haga girar la mezcla a 425 xg durante 10 minutos, y eliminar el sobrenadante. </li><li> Resuspender el precipitado en 8 ml de medio de BMC. Añadir 1 ml de suspensión celular y 9 ml de medio de BMC a 8 bacteriológica placas de Petri. </li><li> Se incuba a 37 ° C, 5% de CO 2. </li><li> El día 5, añadir 10 ml de medio de BMC para cada plato. El día 7, las células se desarrollará plenamente. BMC pueden ser pasados ​​por 1-2 semanas después de la maduración. </li><li> Para dividir BMC, se elimina el medio y añadir 5 ml de PBS frío 1X a cada placa de Petri. Incubar a 4 ° C durante 30 minutos, hasta que las células comienzan a levantar. Enjuague los PMP de la placa con una pipeta de transferencia estéril. </li><li> La piscina del BMC en un tubo cónico de 50 ml y el pellet a 425 xg durante 10 minutos. </li><li> Resuspender el precipitado en 10 ml de medio de BMC y el recuento en un hemocitómetro. Semillas de 2x105 BMC por pocillo en una placa de 24 pocillos con cubreobjetos de vidrio redondas en la parte inferior. Deje que los PMP se adhieran noche a la mañana antes de infectar con T. gondii. BMC en exceso puede ser sembrados en placas de Petri para su uso posterior. </li></ol> 4. Infectar células con T. gondii <ol><li> Infectar a 25 cm 2 frasco de HFFS confluentes con 2x10 6 T. gondii y crecer hasta que las células comienzan a lisar (2-3 días). </li><li> Use un raspador para eliminar células de la monocapa de células huésped infectadas desde el frasco de cultivo de tejido y luego suelte los parásitos de las células huésped pasando la monocapa desalojados a través de una aguja de calibre 27. </li><li> Use un hemocitómetro para determinar el número de parásitos en el medio. </li><li> Infectar a 10 5 parásitos por bien con cubreobjetos de monocapas de HFF del Protocolo n º 1, o de países miembros prestatarios del Protocolo n º 3. Deje que los parásitos invaden durante 3 horas a 37 ° C, 5% de CO 2. </li></ol> 5. Iniciar T. gondii bradizoíto desarrollo por el estrés ambiental <ol><li> Bradizoíto desarrollo en HFFS con el aumento de pH del medio y el hambre de CO 2 <ol class="lalpha"><li> Preparar el medio de desarrollo. Medio de desarrollo contiene RPMI 1640 sin bicarbonato, 1% de SFB, 1% de penicilina-estreptomicina, y 42 mM HEPES. el pH a 8.0 y el filtro de esterilización. </li><li> Quitar DMEM de HFFS infectados, enjuague con PBS 1X, y añadir 1 ml de medio de desarrollo. </li><li> Incubar durante 3 días a 37 º C con el aire ambiente. </li></ol></li><li> La activación de BMC <ol class="lalpha"><li> Preparar el medio para activar la BMC. Medio de la activación es de tamaño medio BMC complementado con IFN-γ 100U/ml y 100 ng / ml LPS. Sonicar LPS en un baño de agua durante 2 minutos antes de su uso para interrumpir los agregados. </li><li> Quitar BMC medio de pozos y reemplazar con 1 ml de medio de activación. </li><li> Se incuba a 37 ° C, 5% de CO 2 durante 3 días. </li></ol></li></ol> 6. Detección de inmunofluorescencia <ol><li> Enjuague infectadas tres veces con PBS 1X. </li><li> Fijar la monocapa con 200ul de 3% de formaldehído durante 20 minutos. </li><li> Retire el fijador y enjuague los pozos con PBS 1X. </li><li> Agregar 200μl de 0,1 M de glicina a los pozos y dejar reposar durante 5 minutos. </li><li> Eliminar la glicina y enjuague los pozos con PBS 1X. </li><li> Añadir 250 ml del 3% BSA/0.2% TritonX-100 en PBS 1X a permeabilizar y bloquear la monocapa durante 30 minutos. </li><li> Retire la solución de bloqueo y enjuagar los pocillos con PBS 1X. </li><li> Añadir 50 l de una dilución 1:250 de rodamina Dolichos biflorus aglutinina en el 3% BSA/0.2% Triton X-100 en PBS 1X. </li><li> Cubierta de la placa y el lugar en la plataforma de agitación a temperatura ambiente durante 1 hora </li><li> Lavar los cubreobjetos 3 veces con 0,2% Triton X-100 en PBS 1X durante 5 minutos cada uno en un agitador de plataforma. </li><li> Cubreobjetos utilizando medio de montaje Vectashield montaje que contiene 4&#39;6-diamidino-2-phenylindole (DAPI). </li><li> Manchado T. gondii pueden ser visualizados con un microscopio de fluorescencia con un filtro apropiado para rodamina con un aumento de 100x. </li></ol> 7. Los resultados representativos La figura 1 muestra tinción representante DBA de T. gondii en BMMs activado y HFFS pH bajo estrés. Ambas manchas DBA mostrar todo parásito que contienen vacuolas, lo que indica la presencia de componentes de la pared del quiste. La imagen muestra la tinción BMM activado DBA que es consistente con la superficie de la vacuola. La sección transversal de la T. gondii en el pH destacó HFFS muestra la pared del quiste sin estructuras internas manchadas. <img src="/files/ftp_upload/2091/2091fig1.jpg" alt="Figura 1" /> Figura 1. DBA destacó la tinción de T. gondii. parásitos intracelulares en condiciones de estrés, BMMs activa o pH destacó HFFS, se tiñeron con DBA rodamina conjugado (rojo). Contraste de interferencia diferencial (DIC) se muestra el esquema del quiste y la barra de escala negro es igual a 2 micras. Los experimentos con animales Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por la Universidad de Wisconsin Cuidado de Animales y el empleo.

<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>

Si bien el mecanismo de desarrollo bradizoíto no se entiende completamente, los análisis de genética molecular de T. conversión gondii etapa de cultivo de tejidos ha llevado al descubrimiento de genes que están involucrados en la formación de quistes bradizoíto 2,3,4. El análisis también dio lugar a la observación de que algunos marcadores bradizoíto se expresan en otras condiciones de estrés prolongado, como el crecimiento en los macrófagos activados 5,6. Los método...

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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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<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>
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<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>
<tr>
<td>RPMI medium 1640 powder (with L-glutamine, without bicarbonate)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>31800-022</td>
<td>&nbsp;</td>
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<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>
<td>&nbsp;</td>		</tbody>
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toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions

Toxoplasma gondii es un parásito intracelular obligado que puede invadir cualquier célula nucleada de animales de sangre caliente. Durante la infección, T. gondii difunde como una forma de replicar rápida llamada de taquizoitos. Taquizoitos de convertirse en una forma enquistada de crecimiento lento llamado bradizoíto por un proceso de señalización que no está bien caracterizado. Dentro de los animales, los quistes bradizoíto se encuentran en el sistema nervioso central y el tejido muscular y representan la etapa crónica de la infección. La conversión a bradizoítos se pueden simular en el cultivo de tejidos de CO 2 de hambre, utilizando un medio con un pH alto, o la adición de interferón gamma (IFN). Bradizoítos se caracterizan por la presencia de una pared del quiste, a la que la lectina de Dolichos biflorus aglutinina (DBA) se une. DBA etiqueta fluorescente se utiliza para visualizar la pared del quiste en los parásitos cultivados en fibroblastos de prepucio humano (HFFS) que han sido expuestos a bajas emisiones de CO 2 y medio de alto pH. Del mismo modo, los parásitos que residen en el hueso murinas derivadas de la médula macrófagos (BMMs) muestran una pared del quiste detectable por DBA después de la BMMs se activan con IFN y lipopolisacárido (LPS). Este protocolo será demostrar cómo inducir a la conversión de T. gondii a bradizoítos utilizando un medio de pH alto crecimiento con bajas emisiones de CO 2 y la activación de BMMs. Las células huésped se cultivaron en cubreobjetos, infectados con taquizoítos y sea activado con la adición de IFN y LPS (BMMs) o expuestos a un medio de pH alto crecimiento (HFFS) de tres días. Al término de las infecciones, las células huésped se fija, permeabilized y bloqueado. Las paredes del quiste se visualizan mediante DBA rodamina con microscopía de fluorescencia.

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 convierte en una forma de quiste en respuesta al estrés ambiental, que puede ser imitado en los modelos de cultivo de tejidos. Este video muestra las técnicas para examinar la formación de la pared del quiste mediante la activación de médula ósea procedentes de los macrófagos o el cambio de pH de crecimiento medio en las células de fibroblastos.

Quiste Toxoplasma gondii Muro de Formación en Activado médula ósea procedentes de los macrófagos y Condiciones bradizoíto

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 vistas.  University of Wisconsin. Toxoplasma gondii se convierte en una forma de quiste en respuesta al estrés ambiental, que puede ser imitado en los modelos de cultivo de tejidos. Este video muestra las técnicas para examinar la formación de la pared del quiste mediante la activación de médula ósea procedentes de los macrófagos o el cambio de pH de crecimiento medio en las células de fibroblastos.

Video: 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

Obtener altamente purificada Toxoplasma gondii Los ooquistes por un gradiente de cloruro de cesio discontinua

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

Investigación

Inmunología e Infección

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.

Una pantalla genética para aislar Toxoplasma gondii Célula huésped-mutantes Egress

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.

Investigación de la polarización de macrófagos utilizando la médula ósea macrófagos derivados

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.

La manipulación genética en<em&gt; Δku80</em&gt; Las cepas de Análisis Genómico Funcional 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.

Médula ósea derivada de macrófagos Producción

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).

Aislamiento y la inyección intravenosa de murino médula ósea derivados monocitos

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.

Adelante Genética pantallas usando macrófagos identificar<em&gt; Toxoplasma gondii</empara la resistencia a IFN-γ por células dependiente&gt; Genes Importante Inmunidad Autónoma

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.

Caracterización metabólica de polarizado M1 y macrófagos derivados de Médula ósea M2 Uso del análisis extracelular Flux en tiempo real

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.

Detección de Inflammasome activación y muerte celular Pyroptotic en macrófagos derivados de médula ósea murinas

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.

Aislamiento y cultivo in vitro de macrófagos derivados de la médula ósea para el estudio de la biología NO-redox

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