עבודה זו נתמכה על ידי אוניברסיטת ויסקונסין במדיסון הרמן א &#39;שפירו נכבדים בוגרת מלגת (CMT), פרס NIH AI072817 (AMP) ו - American Heart Association פרס 0840059N (LJK).

1. הכנת fibroblasts העורלה האדם (HFF) מצופה coverslips <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים coverslip סטרילי זכוכית עגול בחלק התחתון של הבארות של צלחת 24 גם בתרבית רקמה. </li><li style=";text-align:right;direction:rtl"> כדי לקצור HFFs מתוך בקבוקון ומחוברות 2 150cm, לשטוף את הבקבוק פעמיים עם 1X PBS ומוסיפים 2.5 מ&quot;ל של 0.025% טריפסין-EDTA. דגירה בקבוק ב 37 מעלות צלזיוס למשך 5-10 דקות. </li><li style=";text-align:right;direction:rtl"> את הצד הקשה של הבקבוק על כף היד שלך יכול לסייע ניתוק התאים מהבקבוק. לאחר התאים שוחרר מהבקבוק, מוסיפים 150 מ&quot;ל של HFF בינוני (השתנה Dulbecco הנשר בינוני [DMEM] עם 10% FBS, 2 מ&quot;מ L-גלוטמין ו -1% לפניצילין, סטרפטומיצין) ולהשתמש בינוני לשטוף את הבקבוק ולאסוף התאים. </li><li style=";text-align:right;direction:rtl"> לוותר על 1 מ&quot;ל של תאים לכל היטב עם coverslips. </li><li style=";text-align:right;direction:rtl"> אפשר להפוך תאים ומחוברות על 37 מעלות צלזיוס, 5% CO 2. </li></ol> 2. הכנת L929 בינוני מותנה (CM) של BMC פיתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> L929 הוא aneuploid Murine fibrosarcoma תא הקו מפריש המושבה macrophage מגרה גורם לאחר תקופות ארוכות של confluency. הפרשה זו מאפשרת ס&quot;מ L929 התאים לשמש לפיתוח תאים במח העצם עכבר מקרופאגים לתוך התרבות תאים 1. </li><li style=";text-align:right;direction:rtl"> לאחר L929 תאים ומחוברות, לתת להם לדגור על 7-9 ימים נוספים עד שהם מופיעים כדורי ולהתחיל להרים את הבקבוק. איסוף supernatant ו גלולה כל התאים מנותקת XG ב 425 במשך 10 דקות. Supernatant זהו CM. </li><li style=";text-align:right;direction:rtl"> אחת 150 2 ס&quot;מ בקבוק של L929 תאים התשואות 30 מ&quot;ל של CM, אשר מייצר 150 מ&quot;ל של מדיום BMC. BMC בינוני מורכב 10% FBS, CM 20%, 1% פניצילין, סטרפטומיצין, ו -1% ב-L-גלוטמין DMEM. </li></ol> 3. בידוד של מח עצם תרבית תאים של BMCs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להקריב C57BL / 6 העכבר בחנק CO 2 משטח לחטא עם אתנול 70%. </li><li style=";text-align:right;direction:rtl"> לחשוף את הצפק על ידי הרמת עור הבטן ליד הרגל גזירה במספריים. הימנע ניקוב חלל הצפק. צמצמו את הרגל על ​​מנת לחשוף את עצם השוק לבין עצם הירך. גזור רקמת שריר מן העצמות ברגל באמצעות מספריים. </li><li style=";text-align:right;direction:rtl"> כדי להסיר את עצם הירך, חתך פעם מתחת לברך, ופעם ליד הירך. מניחים את העצמות בצלחת פטרי בקטריולוגית המכיל קר 1X PBS. אין להשתמש בתרבית רקמה שטופלו צלחות פטרי על צעדים אלה, משום BMCs תדבק לצמיתות. השתמש באזמל כדי לגרד רקמת שריר נוספת. חותכים את העצם בדיוק מעל הברך כדי לחשוף את מח. </li><li style=";text-align:right;direction:rtl"> חזור על שלבים (ב) ו - (ג) כדי לאסוף את מח העצם של הירך השנייה. </li><li style=";text-align:right;direction:rtl"> השתמש מזרק 10cc מלאים קר 1X PBS ומחט 25 מד כדי לסלק את מח העצם לתוך צינור חרוטי 50 מ&quot;ל. עצם אמור להופיע פעם לבן טהור מוח מוסר. </li><li style=";text-align:right;direction:rtl"> לעבור את התערובת PBS / 22 מח באמצעות מחט מד לשבור את כל אגרגטים מח עצם. </li><li style=";text-align:right;direction:rtl"> ספין את התערובת על XG 425 במשך 10 דקות, ולהסיר את supernatant. </li><li style=";text-align:right;direction:rtl"> Resuspend גלולה ב 8 מ&quot;ל של מדיום BMC. הוסף 1 מ&quot;ל של השעיה התא 9 מ&quot;ל BMC בינוני עד 8 צלחות פטרי בקטריולוגית. </li><li style=";text-align:right;direction:rtl"> לדגור על 37 מעלות צלזיוס, 5% CO 2. </li><li style=";text-align:right;direction:rtl"> ביום 5, להוסיף 10 מ&quot;ל בינוני BMC על כל צלחת. ביום 7, התאים יפותח במלואו. BMCs ניתן passaged במשך 1-2 שבועות לאחר ההבשלה. </li><li style=";text-align:right;direction:rtl"> כדי לפצל BMCs, להסיר את בינוני ומוסיפים 5 מ&quot;ל של קר 1X PBS על כל צלחת פטרי. דגירה על 4 מעלות צלזיוס במשך 30 דקות, עד שהתאים מתחילים להרים. שוטפים את BMCs מהצלחת עם טפטפת העברת סטרילית. </li><li style=";text-align:right;direction:rtl"> בריכת BMCs בצינור חרוטי 50 מ&quot;ל ו גלולה בשעה XG 425 במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> Resuspend גלולה במדיום 10 מ&quot;ל ו - BMC לסמוך hemocytometer. 2x105 Seed BMCs היטב לכל צלחת 24 גם עם coverslips זכוכית עגול על התחתונה. בואו BMCs לדבוק לילה לפני הדבקה עם ט &#39; gondii. BMCs עודף ניתן reseeded על צלחות פטרי לשימוש מאוחר יותר. </li></ol> 4. מדביק תאים עם ט &#39; gondii <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להדביק 25 ס&quot;מ 2 בקבוק של HFFs ומחוברות עם 2x10 6 ט gondii ולגדול עד התאים מתחילים lyse (כ 2-3 ימים). </li><li style=";text-align:right;direction:rtl"> השתמש מגרד תא להסיר את monolayer נגוע התא המארח מהבקבוק בתרבית רקמה מכן שחרר את הטפילים מן התאים לארח על ידי העברת monolayer וחילצה באמצעות מחט 27-מד. </li><li style=";text-align:right;direction:rtl"> השתמש hemocytometer כדי לקבוע את מספר טפילים במדיום. </li><li style=";text-align:right;direction:rtl"> להדביק 10 5 טפילים לכל היטב עם coverslip של HFF monolayers מ 1 לפרוטוקול, או BMCs מן הפרוטוקול 3. בואו הטפילים לפלוש במשך 3 שעות על 37 מעלות צלזיוס, 5% CO 2. </li></ol> 5. ייזום ט gondii bradyzoite פיתוח על ידי לחצים סביבתיים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Bradyzoite פיתוח HFFs עם עלייה pH בינוני 2 CO רעב <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן בינוני פיתוח. פיתוח בינוני מכיל RPMאני 1640 בלי סודה לשתייה, 1% FBS, 1% פניצילין, סטרפטומיצין, ו 42 HEPES מ&quot;מ. pH 8.0 כדי לעקר ולסנן. </li><li style=";text-align:right;direction:rtl"> הסר DMEM מ HFFs נגועים, יש לשטוף עם 1X PBS, ולהוסיף 1ml של התפתחות המדיום. </li><li style=";text-align:right;direction:rtl"> דגירה במשך 3 ימים ב 37 מעלות צלזיוס עם הסביבה האוויר. </li></ol></li><li style=";text-align:right;direction:rtl"> הפעלת BMCs <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן בינוני להפעיל BMCs. בינוני ההפעלה היא מדיום BMC בתוספת 100U/ml IFN-γ ו - 100 ng / ml LPS. Sonicate LPS באמבט מים למשך 2 דקות לפני השימוש כדי לשבש אגרגטים. </li><li style=";text-align:right;direction:rtl"> הסר BMC בינוני מבארות ולהחליף עם 1 מ&quot;ל של מדיום ההפעלה. </li><li style=";text-align:right;direction:rtl"> לדגור על 37 מעלות צלזיוס, 5% CO 2 למשך 3 ימים. </li></ol></li></ol> 6. Immunofluorescence זיהוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשטוף נגוע בארות שלוש פעמים עם 1X PBS. </li><li style=";text-align:right;direction:rtl"> תקן את monolayer עם 200ul של פורמלדהיד 3% למשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> הסר מקבע ולשטוף בארות עם 1X PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 200μl של 0.1 M גליצין לבארות ולתת לשבת במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> הסר גליצין ולשטוף בארות עם 1X PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 250 μl של 3% BSA/0.2% TritonX-100 ב 1X PBS כדי permeabilize ולחסום את monolayer במשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> הסר פתרון חסימת ולשטוף בארות עם 1X PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 50 μl של דילול 1:250 של rhodamine לבלב biflorus agglutinin ב 3% BSA/0.2% Triton X-100 ב 1X PBS. </li><li style=";text-align:right;direction:rtl"> מכסים בצלחת ומניחים על מצע שייקר בטמפרטורת החדר למשך שעה 1 </li><li style=";text-align:right;direction:rtl"> שטפו את coverslips 3 פעמים עם 0.2% Triton X-100 ב 1X PBS במשך 5 דקות כל אחד על שייקר פלטפורמה. </li><li style=";text-align:right;direction:rtl"> Coverslips הר באמצעות מדיום VectaShield הרכבה המכיל 4&#39;6-diamidino-2-phenylindole (DAPI). </li><li style=";text-align:right;direction:rtl"> ויטראז ט gondii ניתן דמיינו באמצעות מיקרוסקופ פלואורסצנטי עם מסנן מתאים עבור rhodamine בהגדלה של 100x. </li></ol> 7. נציג תוצאות איור 1 מציג DBA נציג מכתים של ט &#39; gondii ב BMMs ו HFFs מופעל תחת לחץ pH. שני DBA להראות מכתים סביב טפיל המכיל vacuoles, המעידים על נוכחות של מרכיבים קיר הציסטה. התמונה BMM מופעל מראה מכתים DBA העולה בקנה אחד עם פני השטח של vacuole. חתך של ט &#39; gondii ב-pH הדגיש HFFs מציגה את קיר הציסטה ללא מבנים פנימיים מוכתם. <img src="/files/ftp_upload/2091/2091fig1.jpg" alt="איור 1" /> באיור 1. DBA מכתים של ט הדגיש gondii. טפילים תאיים בתנאי סטרס, BMMs מופעל או pH הדגיש HFFs, הוכתמו DBA מצומדות rhodamine (אדום). הפרעה דיפרנציאלי ניגודיות (DIC) מציגה את קווי המתאר של ציסטה לבין בר סולם שחור שווה 2 מיקרומטר. לניסויים בבעלי חיים ניסויים בבעלי חיים בוצעו בהתאם להנחיות והתקנות שנקבעו על ידי אוניברסיטת ויסקונסין Animal Care ועדת שימוש.

<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>

אין ניגודי אינטרסים הכריז.

בעוד מנגנון הפיתוח bradyzoite לא מובנת לחלוטין, מנתח גנטיקה מולקולרית של ט &#39; המרה gondii הבמה בתרבות רקמה הובילה לגילוי של גנים המעורבים ביצירת ציסטה bradyzoite 2,3,4. מנתח הוביל גם תצפית שכמה סמנים bradyzoite באים לידי ביטוי אחרים בתנאי עקה מתמשך, כולל גידול מקרופאגים ?...

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		<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>
		</table>
		</div>

toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions

Toxoplasma gondii הוא טפיל תאיים לחייב שניתן לפלוש בכל תא גרעיני של דם חם לבעלי חיים. במהלך זיהום, ט gondii מפיצה כמו צורה שכפול מהיר שנקרא tachyzoite. Tachyzoites להמיר לצורה הגדלים לאט encysted שנקרא bradyzoite בתהליך מאותת כי לא מאופיין היטב. בתוך בעלי חיים, ציסטות bradyzoite נמצאים במערכת העצבים המרכזית ורקמות שרירים מייצגים את שלב כרוני של זיהום. הסבת bradyzoites ניתן לדמות בתרבות רקמה ברעב CO 2, באמצעות מדיום עם pH גבוה, או תוספת של אינטרפרון גאמה (IFNγ). Bradyzoites מאופיינים על ידי נוכחות של קיר הציסטה, אשר לקטינים לבלב biflorus agglutinin (DBA) נקשר. DBA שכותרתו fluorescently משמש כדי להמחיש את קיר הציסטה ב טפילים הגדלים fibroblasts העורלה האדם (HFFs) כי נחשפו CO 2 נמוך ובינוני pH גבוה. באופן דומה, טפילים המתגוררים מח עצם Murine הנגזרות מקרופאגים (BMMs) להציג קיר הציסטה לזיהוי על ידי DBA לאחר BMMs מופעלים עם IFNγ ו lipopolysaccharide (LPS). פרוטוקול זה יהיה להדגים כיצד לגרום ההמרה של ט &#39; gondii כדי bradyzoites באמצעות מדיום הגידול pH גבוה עם CO 2 הפעלה נמוך של BMMs. תאים מארח יהיה מתורבת על coverslips, נגוע tachyzoites והופעל גם עם תוספת של IFNγ ו LPS (BMMs) או חשיפה בינונית צמיחה גבוה pH (HFFs) במשך שלושה ימים. עם השלמת זיהומים, התאים המארחים יהיה קבוע, permeabilized, וחסמו. קירות הציסטה יהיה דמיינו באמצעות DBA rhodamine עם מיקרוסקופ פלואורסצנטי.

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 ממיר כדי ליצור ציסטה בתגובה מדגיש סביבתיים, אשר ניתן חיקה במודלים של תרביות רקמה. וידאו זה מדגים טכניקות כדי לבחון הקמת קיר הציסטה על ידי הפעלת מח עצם הנגזרות מקרופאגים או שינוי בינוני צמיחה pH בתאים פיברובלסטים.

Toxoplasma וול gondii ציסטה כינונה ב הופעל מח עצם הנגזרות המקרופאגים והגבלות 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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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 ממיר כדי ליצור ציסטה בתגובה מדגיש סביבתיים, אשר ניתן חיקה במודלים של תרביות רקמה. וידאו זה מדגים טכניקות כדי לבחון הקמת קיר הציסטה על ידי הפעלת מח עצם הנגזרות מקרופאגים או שינוי בינוני צמיחה pH בתאים פיברובלסטים.

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

קבלת מטוהרים Toxoplasma gondii שביצים ידי Gradient כלוריד רציפה וצזיום

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

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.

מסך גנטית לבודד Toxoplasma gondii מארח תאים יציאה מוטציות

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.

חקירה של קיטוב Macrophage שימוש המקרופאגים נגזרים מח עצם

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.

מניפולציה גנטית ב<em&gt; Δku80</em&gt; זנים לאנליזה גנומית תפקודית של<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.

מח עצם נגזרות Macrophage הפקה

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).

המונוציטים בידוד והזרקה תוך ורידית של Murine מח העצם נגזר

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.

קדימה גנטיקה מסכי שימוש המקרופאגים לזיהוי<em&gt; Toxoplasma gondii</emחסינות אוטונומית נייד להתנגדות לIFN-γ-Dependent&gt; גנים חשובים

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.

אפיון חילוף חומרים של מקוטב M1 והמקרופאגים נגזר מח עצם M2 באמצעות ניתוח תאי שטף בזמן אמת

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.

זיהוי של Inflammasome ההפעלה ומוות תא Pyroptotic של מקרופאגים הנגזרות מח עצם מאתר

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.

בידוד ותרבית במבחנה של מקרופאגים שמקורם במח עצם לחקר הביולוגיה של NO-Redox

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