Diese Arbeit wurde von der University of Wisconsin-Madison Herman I. Shapiro Distinguished Graduate Fellowship (CMT), NIH Auszeichnung AI072817 (AMP) und American Heart Association Award 0840059N (LJK) unterstützt.

1. Vorbereitung der menschlichen Vorhaut-Fibroblasten (HFF)-beschichteten Deckgläschen <ol><li> Legen Sie eine sterile runde Deckglas auf dem Boden der Wells einer 24-well Zellkulturschale. </li><li> Zur Ernte HFFS aus einer konfluenten 150cm 2 Flasche, spülen Sie die Flasche zweimal mit 1X PBS und 2,5 ml von 0,025% Trypsin-EDTA. Inkubieren Kolben bei 37 ° C für 5-10 Minuten. </li><li> Tippen Sie auf die Seite der Kolben gegen die Handfläche in Ablösen der Zellen aus der Flasche zu unterstützen. Sobald Zellen aus dem Kolben gelöst, fügen Sie 150 ml der HFF Medium (Dulbecco Modified Eagle Medium [DMEM] mit 10% FBS, 2 mM L-Glutamin und 1% Penicillin-Streptomycin) zu verwenden und das Medium in den Kolben spülen und zu sammeln der Zellen. </li><li> Dispense 1 ml der Zellen pro Vertiefung mit dem Deckgläschen. </li><li> Lassen Zellen zu bei 37 konfluenten ° C, 5% CO 2. </li></ol> 2. Vorbereitung L929 konditioniertem Medium (CM) für BMC Entwicklung <ol><li> L929 ist eine murine aneuploid Fibrosarkom-Zelllinie, die Makrophagen-Kolonie-stimulierender Faktor sondert nach längerer Konfluenz. Dieses Sekret kann der CM von L929-Zellen verwendet werden, um auf Knochenmarkszellen der Maus in die Makrophagen in Zellkultur 1 zu entwickeln sein. </li><li> Sobald L929 Zellen konfluent sind, lassen Sie sie für eine weitere 7-9 Tage inkubieren, bis sie kugelförmig und erscheinen beginnen Abheben des Kolbens. Sammeln Überstand und Pellet keine abgelösten Zellen bei 425 xg für 10 Minuten. Dieser Überstand ist der CM. </li><li> Ein 150 cm 2-Kolben von L929-Zellen ergibt 30 ml CM, die 150 ml des BMC Medium erzeugt. BMC Medium ist von 10% FBS, 20% CM, 1% Penicillin-Streptomycin und 1% L-Glutamin in DMEM zusammen. </li></ol> 3. Isolierung von Knochenmark-und Zellkultur von BMC <ol><li> Opfern C57BL / 6 Maus durch CO 2 Ersticken und Oberfläche sterilisieren mit 70% Ethanol. </li><li> Expose des Bauchfells durch Anheben der Bauchhaut in der Nähe des Beines und Schneiden mit der Schere. Vermeiden Sie Punktion der Bauchhöhle. Reduzieren Sie das Bein an der Tibia und des Femurs freizulegen. Cut Muskelgewebe aus dem Beinknochen mit einer Schere. </li><li> Um den Oberschenkelknochen entfernen, einmal unter dem Knie und einmal in der Nähe der Hüfte geschnitten. Legen Sie die Knochen in eine bakteriologische Petrischale mit kaltem 1X PBS. Verwenden Sie keine Gewebekultur behandelt Petrischalen für diese Schritte, da die BMC dauerhaft halten wird. Verwenden Sie ein Skalpell abkratzen zusätzliche Muskelgewebe. Schneiden Sie die Knochen knapp oberhalb des Knies bis auf die Knochen freizulegen. </li><li> Wiederholen Sie die Schritte (b) und (c) des Knochenmarks aus der zweiten Femur zu sammeln. </li><li> Verwenden Sie einen 10cc Spritze mit kaltem 1X PBS und eine 25-Gauge-Nadel in das Knochenmark in einem 50 ml konischen Rohr entfernen gefüllt. Die Knochen erscheinen soll reinweiß einmal Knochenmark entnommen wird. </li><li> Führen Sie das PBS / Knochenmark-Gemisch durch eine 22-Gauge-Nadel zu zerbrechen alle Knochenmark-Aggregate. </li><li> Drehen Sie die Mischung bei 425 xg für 10 Minuten, und entfernen Sie den Überstand. </li><li> Das Pellet in 8 ml BMC Medium. 1 ml der Zellsuspension und 9 ml BMC Medium zu 8 bakteriologischen Petrischalen. </li><li> Bei 37 ° C, 5% CO 2. </li><li> Am Tag 5, 10 ml BMC Medium zu jeder Platte. Am Tag 7, werden die Zellen voll entwickelt sein. BMC können für 1-2 Wochen nach der Reifung passagiert werden. </li><li> So teilen BMC, entfernen Sie das Medium und mit 5 ml kaltem 1X PBS, um jede Petrischale. Inkubation bei 4 ° C für 30 Minuten, bis die Zellen zu heben beginnen. Spülen Sie den BMC von der Platte mit einem sterilen Transferpipette. </li><li> Pool der BMC in einem 50 ml konischen Rohr-und Pellet bei 425 xg für 10 Minuten. </li><li> Das Pellet in 10 ml Medium BMC und zählen auf einer Zählkammer. Seed 2x105 BMC pro Well in einer 24-Well-Platte mit runden Deckgläschen auf der Unterseite. Lassen Sie den BMC über Nacht halten, bevor Infektion mit T. gondii. Excess BMC kann auf Petrischalen für die spätere Verwendung reseeded werden. </li></ol> 4. Infizieren Zellen mit T. gondii <ol><li> Infect 25 cm 2 Flasche konfluent HFFS mit 2x10 6 T. gondii und wachsen, bis die Zellen zu lysieren beginnen (ca. 2-3 Tage). </li><li> Verwenden Sie einen Zellschaber der infizierten Wirtszelle Monoschicht aus der Zellkulturflasche entfernen Sie dann die Parasiten aus den Wirtszellen, indem das verdrängt Monoschicht durch eine 27-Gauge-Nadel. </li><li> Verwenden Sie eine Zählkammer die Anzahl der Parasiten in das Medium zu bestimmen. </li><li> Infect 10 5 Parasiten pro Well mit Deckglas des HFF-Monoschichten von Protokoll 1, oder BMC von Protokoll Nr. 3. Lassen Sie die Parasiten dringen 3 Stunden bei 37 ° C, 5% CO 2. </li></ol> 5. Initiieren von T. gondii bradyzoite Entwicklung von Belastungen der Umwelt <ol><li> Bradyzoite Entwicklung in HFFS mit mittleren pH-Wert erhöhen und die CO 2 Hungertod <ol class="lalpha"><li> Bereiten Entwicklung Medium. Entwicklung Medium enthält RPMI 1640 ohne Bicarbonat, 1% FBS, 1% Penicillin-Streptomycin und 42 mM HEPES. pH auf 8,0 und Filter zu sterilisieren. </li><li> Entfernen DMEM aus infizierten HFFS mit 1X PBS spülen, und fügen Sie 1ml Entwicklung Medium. </li><li> Inkubieren für 3 Tage bei 37 ° C mit Umgebungsluft. </li></ol></li><li> Die Aktivierung von BMC <ol class="lalpha"><li> Bereiten Sie mittel-bis BMC aktivieren. Activation Medium ist BMC Medium mit IFN-γ 100U/ml und 100 ng / ml LPS ergänzt. Ultraschallbad LPS in einem Wasserbad für 2 Minuten vor der Verwendung zu Aggregaten zu stören. </li><li> Entfernen BMC Medium aus Brunnen und ersetzen mit 1 ml der Aktivierung Medium. </li><li> Bei 37 ° C, 5% CO 2 für 3 Tage. </li></ol></li></ol> 6. Immunfluoreszenz-Nachweis <ol><li> Spülen infizierten Vertiefungen dreimal mit 1X PBS. </li><li> Fix der Monoschicht mit 200 &amp;mgr; l von 3% Formaldehyd für 20 Minuten. </li><li> Entfernen Fixativ und spülen Brunnen mit 1X PBS. </li><li> Add 200 ul von 0,1 M Glycin, um Brunnen und lassen Sie sich für 5 Minuten. </li><li> Entfernen Glycin und spülen Brunnen mit 1X PBS. </li><li> Add 250 ul 3% BSA/0.2% TritonX-100 in 1X PBS permeabilisiert und blockieren die Monolayer für 30 Minuten. </li><li> Entfernen Sie blockiert und spülen Brunnen mit 1X PBS. </li><li> Add 50 ul einer 1:250 Verdünnung von Rhodamin Dolichos biflorus Agglutinin in 3% BSA/0.2% Triton X-100 in 1X PBS. </li><li> Abdeckplatte und legen auf der Plattform Schüttler bei Raumtemperatur für 1 Stunde </li><li> Waschen Sie die Deckgläschen 3 mal mit 0,2% Triton X-100 in 1X PBS für jeweils 5 Minuten auf einer Plattform Schüttler. </li><li> Berg Deckgläser mit Vectashield Eindeckmedium mit 4&#39;6-diamidino-2-phenylindole (DAPI). </li><li> Stained T. gondii visualisiert werden können mit Hilfe eines Fluoreszenz-Mikroskops mit einem Filter geeignet für Rhodamin bei einer Vergrößerung von 100x. </li></ol> 7. Repräsentative Ergebnisse Abbildung 1 zeigt repräsentative DBA-Färbung von T. gondii in aktiviert BMMS und HFFS unter pH Stress. Beide zeigen DBA Färbung um Parasiten mit Vakuolen, die das Vorhandensein von Zystenwand Komponenten. Die aktivierte BMM Bild zeigt DBA Färbung, die mit der Oberfläche der Vakuole ist. Der Querschnitt des T. gondii in pH betonte HFFS zeigt die Zystenwand ohne internen Strukturen gefärbt. <img src="/files/ftp_upload/2091/2091fig1.jpg" alt="Abbildung 1" /> Abbildung 1. DBA Färbung betont T. gondii. intrazelluläre Parasiten unter Stress-Bedingungen, aktiviert BMMS oder pH betonte HFFS wurden mit Rhodamin konjugiert DBA (rot) gefärbt. Differential-Interferenz-Kontrast (DIC) zeigt den Umriss der Zyste und dem schwarzen Maßstab entspricht 2 pM. Experimente an Tieren Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von der University of Wisconsin Animal Care und Verwenden Ausschusses durchgeführt.

<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>

Während der Mechanismus der bradyzoite Entwicklung ist noch nicht vollständig verstanden, molekulargenetische Analysen von T. gondii Bühne Umwandlung in Gewebekultur hat zur Entdeckung von Genen, die in bradyzoite Zystenbildung 2,3,4 beteiligt sind geführt. Die Analysen auch auf die Beobachtung, dass einige bradyzoite Marker in anderen längeren Stress-Bedingungen, einschließlich des Wachstums in aktivierten Makrophagen 5,6 ausgedrückt werden geführt. Die oben genannten Methoden bes...

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		<thead>
		<tr>
		<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 ist ein obligat intrazelluläre Parasiten, die jeder kernhaltigen Zelle des Warmblüter eindringen können. Während der Infektion, T. gondii verbreitet als schneller replizierende Form genannt tachyzoite. Tachyzoiten in eine langsam wachsende encystierten Form genannt bradyzoite durch eine Signalisierung, die nicht gut charakterisiert konvertieren. Innerhalb Tiere sind bradyzoite Zysten in das zentrale Nervensystem und Muskelgewebe und stellen die chronische Phase der Infektion. Die Umstellung auf Bradyzoiten können in Gewebekultur simuliert CO 2 Hunger, mit Medium mit hoher pH-Wert oder die Zugabe von Interferon-gamma (IFN &amp;ggr;). Bradyzoiten werden durch die Anwesenheit einer Zystenwand, zu denen das Lektin Dolichos biflorus Agglutinin (DBA) bindet charakterisiert. Fluoreszenzmarkierten DBA wird die Zystenwand in Parasiten im menschlichen Vorhaut-Fibroblasten (HFFS), die zu niedrigen CO 2 und hoher pH-Milieu ausgesetzt waren, gewachsen zu visualisieren. Ebenso, wohnhaft Parasiten in murinen Knochenmark-Makrophagen (BMMS) zeigt eine Zystenwand nachweisbar durch DBA nach dem BMMS mit IFNy und Lipopolysaccharid (LPS) aktiviert werden. Dieses Protokoll wird zeigen, wie die Umwandlung von T. induzieren gondii zu Bradyzoiten mit einem hohen pH-Wachstumsmedium mit niedrigem CO 2 und die Aktivierung der BMMS. Wirtszellen wird auf Deckgläsern kultiviert werden, infiziert mit Tachyzoiten und entweder mit Zugabe von IFN &amp;ggr; und LPS (BMMS) aktiviert oder die von einem hohen pH Wachstumsmedium (HFFS) für drei Tage. Nach Abschluss der Infektionen wird Wirtszellen fixiert, permeabilisiert werden, und blockiert. Cyst Wände werden visualisiert Rhodamin DBA mit Fluoreszenzmikroskopie.

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 verwandelt sich in eine Zyste bilden als Reaktion auf Belastungen der Umwelt, die in Gewebekultur-Modellen nachgeahmt werden kann. Dieses Video zeigt Techniken, um Zystenwand Bildung durch Aktivierung des Knochenmarks-Makrophagen oder Ändern Wachstumsmedium pH in Fibroblasten-Zellen zu untersuchen.

Toxoplasma gondii Zystenwand Formation in Activated Knochenmark abgeleiteten Makrophagen und Bradyzoite AGB

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 Aufrufe.  University of Wisconsin. Toxoplasma gondii verwandelt sich in eine Zyste bilden als Reaktion auf Belastungen der Umwelt, die in Gewebekultur-Modellen nachgeahmt werden kann. Dieses Video zeigt Techniken, um Zystenwand Bildung durch Aktivierung des Knochenmarks-Makrophagen oder Ändern Wachstumsmedium pH in Fibroblasten-Zellen zu untersuchen.

Serie: 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

Gewinnung von hochgradig gereinigter Toxoplasma gondii Oocysten durch eine diskontinuierliche Cäsiumchloridgradienten

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

Forschung

Immunologie und Infektion

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.

Ein genetischer Screen zu isolieren Toxoplasma gondii Host-Zelle Egress Mutanten

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.

Untersuchung von Makrophagen-Polarisation mit Knochenmark-Makrophagen

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.

Genetische Manipulation in<em&gt; Δku80</em&gt; Die Stämme für Funktionelle Genomanalyse<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.

Knochenmark-abgeleiteten Makrophagen-Produktion

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).

Isolierung und intravenöse Injektion von Maus-Knochenmark stammenden Monozyten

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.

Zukunfts Genetics Screens Mit Makrophagen zu identifizieren<em&gt; Toxoplasma gondii</em&gt; Gene wichtig für die Resistenz gegenüber IFN-γ-abhängige Zell autonome Immunität

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.

Metabolische Charakterisierung Polarized M1 und M2-Knochenmark-Makrophagen über die Echtzeit-Extrazelluläre Flußanalyse

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.

Erkennung von Inflammasome Aktivierung und Pyroptotic Zelltod in murinen Makrophagen Knochenmark abgeleitet

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.

Isolierung und In vitro Kultur von Knochenmark-abgeleiteten Makrophagen für das Studium der NO-Redox-Biologie

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