Questo lavoro è stato sostenuto dalla University of Wisconsin-Madison Herman I. Shapiro Distinguished Graduate Fellowship (CMT), premio NIH AI072817 (AMP) e dell&#39;American Heart Association Award 0840059N (LJK).

1. Preparazione dei fibroblasti prepuzio umano (HFF) rivestite coprioggetto <ol><li> Mettere un coprioggetti sterile di vetro rotonda sul fondo dei pozzetti di una 24-pozzetti di coltura tissutale. </li><li> Per raccogliere HFFS da un confluente 150 centimetri 2 fiasco, lavare il pallone due volte con PBS 1X e aggiungere 2,5 ml di 0,025% tripsina-EDTA. Incubare Pallone a 37 ° C per 5-10 minuti. </li><li> Toccando il lato del pallone contro il palmo della mano può aiutare a staccare le cellule dal pallone. Una volta che le cellule hanno liberato dal pallone, aggiungere 150 ml di HFF media (Dulbecco Modified Aquila Media [DMEM] con 10% FBS, 2 mM L-glutamina e 1% di penicillina-streptomicina) e utilizzare il mezzo per sciacquare il pallone e raccogliere le cellule. </li><li> Dispensare 1 ml di cellule per bene con i coprioggetti. </li><li> Consentono alle cellule di diventare confluenti a 37 ° C, 5% di CO 2. </li></ol> 2. Preparazione L929 mezzo condizionato (CM) per lo sviluppo BMC <ol><li> L929 è un fibrosarcoma murino linea di cellule aneuploidi che secerne il fattore stimolante colonie di macrofagi dopo lunghi periodi di confluenza. Questa secrezione permette al CM da L929 cellule da utilizzare per sviluppare il mouse cellule del midollo osseo in macrofagi in coltura cellulare 1. </li><li> Una volta L929 cellule sono confluenti, lasciarli incubare per altri 7-9 giorni fino a che non appaiono sferici e iniziare a sollevare fuori il pallone. Raccogliere il supernatante e pellet eventuali cellule staccato a 425 xg per 10 minuti. Questo è il surnatante CM. </li><li> Uno a 150 cm 2 fiasco di cellule L929 rendimenti 30 ml di CM, che genera 150 ml di medium BMC. BMC mezzo è composto da 10% FBS, 20% CM, 1% di penicillina-streptomicina e 1% di L-glutamina in DMEM. </li></ol> 3. Isolamento del midollo osseo e di colture cellulari di BMC <ol><li> Sacrificare uno C57BL / 6 del mouse asfissia da CO 2 e la superficie sterilizzare con il 70% di etanolo. </li><li> Esporre il peritoneo sollevando la cute addominale vicino alla gamba e il taglio con le forbici. Evitare la puntura della cavità peritoneale. Ridurre alla gamba per esporre la tibia ed il femore. Tagliare il tessuto muscolare dalle ossa delle gambe con le forbici. </li><li> Per rimuovere il femore, una volta tagliata sotto il ginocchio e una volta vicino al fianco. Mettere le ossa in un piatto batteriologico Petri contenenti freddo PBS 1X. Non utilizzare colture di tessuti trattati piastre di Petri per questi passi perché il BMC aderisce in modo permanente. Utilizzare un bisturi per raschiare il tessuto muscolare in più. Tagliare l&#39;osso appena sopra il ginocchio per esporre il midollo. </li><li> Ripetere i punti da (b) e (c) per raccogliere midollo osseo da femore secondo. </li><li> Utilizzare una siringa 10cc riempito con PBS 1X freddo e da 25 gauge per rimuovere il midollo osseo in una provetta da 50 ml. L&#39;osso deve apparire puro midollo bianco una volta è stato rimosso. </li><li> Passare il composto PBS / midollo attraverso un ago 22 gauge per rompere ogni aggregati midollo osseo. </li><li> Spin la miscela a 425 xg per 10 minuti, e rimuovere il surnatante. </li><li> Risospendere il pellet in 8 ml di terreno BMC. Aggiungere 1 ml di sospensione cellulare e 9 ml di medie BMC a 8 piatti batteriologico Petri. </li><li> Incubare a 37 ° C, 5% di CO 2. </li><li> Il giorno 5, aggiungere 10 ml di media BMC per ogni piatto. Al 7 ° giorno, le cellule saranno pienamente sviluppati. BMC possono essere diversi passaggi per 1-2 settimane dopo la maturazione. </li><li> Per dividere BMC, rimuovere il supporto e aggiungere 5 ml di PBS 1X freddo ad ogni piastra di Petri. Incubare a 4 ° C per 30 minuti, fino a quando le cellule cominciano a sollevare. Sciacquare la BMC via il piatto con una pipetta sterile per il trasferimento. </li><li> Piscina del BMC in un tubo da 50 ml conica e pellet a 425 xg per 10 minuti. </li><li> Risospendere il pellet in 10 ml di mezzo di BMC e contare su un emocitometro. Seme 2x105 BMC per bene in un 24-pozzetti con coprioggetto di vetro rotondo sul fondo. Lasciate che il BMC aderire durante la notte prima di infettare con T. gondii. BMC in eccesso può essere reseeded su piastre di Petri per un uso successivo. </li></ol> 4. Infettare le cellule con T. gondii <ol><li> Infettare 25 cm 2 fiasco di HFFS confluenti con 2x10 6 T. gondii e crescere fino a quando le cellule iniziano a lisare (circa 2-3 giorni). </li><li> Utilizzare un raschietto per rimuovere le cellule infette monostrato cellula ospite dal pallone di coltura tissutale quindi rilasciare i parassiti dalle cellule ospiti passando il monostrato sloggiato con una 27-gauge. </li><li> Utilizzare un emocitometro per determinare il numero di parassiti nel terreno. </li><li> Infettare 10 5 parassiti per bene con coprioggetto di monostrati HFF dal protocollo n. 1, o BMC dal protocollo 3. Lasciate che i parassiti invadono per 3 ore a 37 ° C, 5% di CO 2. </li></ol> 5. Avvio T. gondii sviluppo bradyzoite da stress ambientali <ol><li> Sviluppo Bradyzoite in HFFS con aumento del pH medio e CO 2 fame <ol class="lalpha"><li> Preparare di sviluppo a medio. Di sviluppo a medio contiene RPMI 1640 senza bicarbonato, 1% FBS, 1% di penicillina-streptomicina, e 42 HEPES mM. pH a 8,0 e filtrare sterilizzare. </li><li> Rimuovere DMEM da HFFS infetti, sciacquare con PBS 1X, e aggiungere 1 ml di sviluppo a medio. </li><li> Incubare per 3 giorni a 37 ° C con aria ambiente. </li></ol></li><li> Attivazione di BMC <ol class="lalpha"><li> Preparare medio per attivare BMC. Medio di attivazione è di media BMC integrato con 100U/ml IFN-γ e 100 ng / ml LPS. Sonicare LPS in un bagno d&#39;acqua per 2 minuti prima dell&#39;uso per interrompere aggregati. </li><li> Rimuovere BMC media da pozzi e sostituirlo con 1 ml di terreno di attivazione. </li><li> Incubare a 37 ° C, 5% di CO 2 per 3 giorni. </li></ol></li></ol> 6. Rilevamento immunofluorescenza <ol><li> Sciacquare infetti pozzi tre volte con PBS 1X. </li><li> Fissare il monostrato con 200ul del 3% di formaldeide per 20 minuti. </li><li> Rimuovere fissativo e risciacquare pozzetti con PBS 1X. </li><li> Aggiungere 200μl di 0,1 M glicina a pozzi e lasciate riposare per 5 minuti. </li><li> Rimuovere glicina e risciacquare pozzetti con PBS 1X. </li><li> Aggiungere 250 microlitri del 3% BSA/0.2% TritonX-100 in PBS 1X per permeabilize e bloccare il monostrato per 30 minuti. </li><li> Rimuovere soluzione di saturazione e risciacquare pozzetti con PBS 1X. </li><li> Aggiungere 50 ml di una diluizione 1:250 di rodamina Dolichos biflorus agglutinina nel 3% BSA/0.2% Triton X-100 in PBS 1X. </li><li> Piastra di copertura e posto su piattaforma agitatore a temperatura ambiente per 1 ora </li><li> Lavare i coprioggetti 3 volte con 0,2% Triton X-100 in PBS 1X per 5 minuti ciascuno su un agitatore piattaforma. </li><li> Coprioggetto montare utilizzando medium Vectashield montaggio contenente 4&#39;6-diamidino-2-fenilindolo (DAPI). </li><li> Macchiato T. gondii può essere visualizzato usando un microscopio a fluorescenza con un appropriato filtro per rodamina ad un ingrandimento di 100x. </li></ol> 7. Rappresentante risultati La figura 1 mostra DBA colorazione rappresentante della T. gondii in BMMs attivato e HFFS sotto stress pH. Sia le macchie DBA spettacolo intorno parassita contenente vacuoli, che indica la presenza di componenti di parete cistica. L&#39;immagine mostra BMM attivato colorazione DBA che è coerente con la superficie del vacuolo. La sezione di T. gondii del pH sottolineato HFFS mostra la parete cistica senza strutture interne macchiato. <img src="/files/ftp_upload/2091/2091fig1.jpg" alt="Figura 1" /> Figura 1. Colorazione di DBA sottolineato T. gondii. parassiti intracellulari in condizioni di stress, BMMs attivato o pH sottolineato HFFS, sono stati marcati con rodamina DBA coniugato (rosso). Contrasto interferenziale differenziale (DIC) mostra la sagoma della cisti e la barra di scala nera è uguale a 2 micron. Esperimenti su animali Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dalla University of Wisconsin Cura degli animali e del Comitato Usa.

<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>

Nessun conflitto di interessi dichiarati.

Mentre il meccanismo di sviluppo bradyzoite non è pienamente compreso, analisi di genetica molecolare di T. stadio di conversione gondii in colture di tessuti ha portato alla scoperta di geni che sono coinvolti nella formazione di cisti bradyzoite 2,3,4. Le analisi inoltre ha portato alla constatazione che alcuni marcatori bradyzoite sono espressi in altre condizioni di stress prolungato, tra cui la crescita in macrofagi attivati ​​5,6. I metodi sopra descrivono come far cres...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<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>
<tr>
<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>
<tr>
<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>
<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>
<td>&nbsp;</td>		</tbody>
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toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions

Toxoplasma gondii è un parassita intracellulare obbligato che possono infettare ogni cellula nucleata di animali a sangue caldo. Durante l&#39;infezione, T. gondii diffonde come una forma veloce replicando chiamato tachizoite. Tachizoiti convertire in una lenta crescita forma incistata chiamato bradyzoite da un processo di segnalazione che non è ben caratterizzato. All&#39;interno di animali, cisti bradyzoite si trovano nel sistema nervoso centrale e del tessuto muscolare e rappresentano la fase cronica di infezione. Conversione in bradyzoites può essere simulato in coltura da CO 2 fame, utilizzando medio alta con un pH, o l&#39;aggiunta di interferone gamma (IFNγ). Bradyzoites sono caratterizzate dalla presenza di un muro di cisti, a cui la lectina Dolichos biflorus agglutinina (DBA) si lega. DBA fluorescente viene utilizzato per visualizzare le cisti di parassiti muro cresciuto in fibroblasti umani prepuzio (HFFS) che sono stati esposti a basse emissioni di CO 2 e medio alto pH. Allo stesso modo, i parassiti che risiedono nel midollo osseo murino di derivazione macrofagi (BMMs) visualizzare una parete cisti rilevabile dal DBA dopo la BMMs sono attivate con IFNγ e lipopolisaccaride (LPS). Questo protocollo verrà illustrato come indurre la conversione di T. gondii a bradyzoites utilizzando un mezzo di crescita elevati pH con 2 CO bassa e l&#39;attivazione di BMMs. Cellule ospiti saranno coltivati ​​su vetrini, infettati con tachizoiti e sia attivato con aggiunta di IFNγ e LPS (BMMs) o esposti a una media di crescita elevato pH (HFFS) per tre giorni. Al termine delle infezioni, cellule ospiti sarà fissato, permeabilizzate, e bloccato. Pareti cisti verrà visualizzato usando DBA rodamina con microscopia a fluorescenza.

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 converte in una forma cisti in risposta a stress ambientali, che può essere imitato in modelli di colture di tessuti. Questo video mostra le tecniche per esaminare la formazione di cisti muro attivando midollo osseo macrofagi o modificare il pH medio di crescita in cellule di fibroblasti.

Toxoplasma gondii parete formazione di cisti in attivo dal midollo osseo macrofagi e condizioni 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

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JoVE Journal

Immunology and Infection

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

16.1K Views.  University of Wisconsin. Toxoplasma gondii converte in una forma cisti in risposta a stress ambientali, che può essere imitato in modelli di colture di tessuti. Questo video mostra le tecniche per esaminare la formazione di cisti muro attivando midollo osseo macrofagi o modificare il pH medio di crescita in cellule di fibroblasti.

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

Ottenere altamente purificato Toxoplasma gondii Oocisti da un gradiente discontinuo di cloruro di cesio

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

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Immunologia e infezioni

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.

Uno screening genetico per isolare Toxoplasma gondii Host-cell Egress Mutants

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.

Indagine di polarizzazione dei macrofagi Utilizzando Bone Marrow macrofagi derivati

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 manipolazione genetica in<em&gt; Δku80</em&gt; Ceppi per analisi genomica funzionale di<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.

Derivate dal midollo osseo macrofagi 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).

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

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 Genetica schermi usando macrofagi per identificare<em&gt; Toxoplasma gondii</em&gt; geni importanti per la Resistenza di IFN-γ-Dependent cellulare Immunità autonoma

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.

Caratterizzazione metabolica di Polarized M1 e M2 macrofagi derivate dal midollo osseo Utilizzando analisi in tempo reale extracellulare 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.

Rilevamento di attivazione Inflammasome e morte delle cellule Pyroptotic in macrofagi murini derivate da midollo osseo

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.

Isolamento e coltura in vitro di macrofagi derivati dal midollo osseo per lo studio della biologia NO-Redox

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