وأيد هذا العمل من قبل جامعة ويسكونسن ماديسون هيرمان أولا شابيرو زمالة دراسات عليا المميز (CMT) ، المعاهد الوطنية للصحة جائزة AI072817 (AMP) ، والاميركي جائزة جمعية القلب 0840059N (LJK).

1. إعداد الإنسان الليفية القلفة (HFF) المغلفة coverslips <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان معقم ساترة زجاجية مستديرة حول الجزء السفلي من الآبار لوحة نسيج الثقافة 24 أيضا. </li><li style=";text-align:right;direction:rtl"> لحصاد HFFs من قارورة 2 150cm متموجة ، وشطف القارورة مرتين مع 1X PBS وإضافة 2.5 مل من 0.025 ٪ التربسين - EDTA. احتضان القارورة عند 37 درجة مئوية لمدة 5-10 دقائق. </li><li style=";text-align:right;direction:rtl"> ويمكن الاستفادة من الجانب القارورة ضد كف يدك المساعدات في فصل الخلايا من القارورة. مرة واحدة الخلايا قد أفرج عنه من القارورة ، إضافة 150 مل من المتوسط ​​HFF (Dulbecco في التعديل النسر متوسطة [DMEM] مع FBS 10 ٪ ، 2 مم L - الجلوتامين ، و 1 ٪ البنسلين الستربتوميسين) واستخدام وسيلة لغسل القارورة وجمع الخلايا. </li><li style=";text-align:right;direction:rtl"> الاستغناء 1 مل من الخلايا لكل بئر مع coverslips. </li><li style=";text-align:right;direction:rtl"> السماح لتصبح خلايا متكدسة عند 37 درجة مئوية ، 5 ٪ CO 2. </li></ol> 2. إعداد L929 المتوسطة مكيفة (CM) لتطوير BMC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> L929 هو مختل الصيغة الصبغية ليفية الفئران خط الخلية التي تفرز عامل تحفيز مستعمرة بلعم بعد فترات طويلة من confluency. هذا إفراز تمكن من L929 CM الخلايا لاستخدامها لتطوير خلايا فأر في نخاع العظم الضامة في الثقافة الخلية 1. </li><li style=";text-align:right;direction:rtl"> مرة واحدة L929 الخلايا متموجة ، والسماح لهم لاحتضان أيام 7-9 اضافية حتى تظهر كروية وبدء رفع قبالة القارورة. جمع وطاف بيليه أية خلايا منفصلة في 425 x ج لمدة 10 دقائق. هذا هو طاف CM. </li><li style=";text-align:right;direction:rtl"> واحد 150 سم 2 قارورة من الخلايا L929 غلة 30 مل من CM ، الذي يولد 150 مل من المتوسط ​​BMC. وتتألف من FBS BMC المتوسطة 10 ٪ ، CM 20 ٪ ، 1 ٪ البنسلين الستربتوميسين ، و 1 ٪ في الجلوتامين L - DMEM. </li></ol> 3. العزلة من نخاع العظم والثقافة خلية من خلايا نخاع العظام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> التضحية C57BL 6 / 2 الماوس خنقا أكسيد الكربون وتعقيم السطح مع الايثانول 70 ٪. </li><li style=";text-align:right;direction:rtl"> فضح البريتوني من خلال رفع جلد البطن بالقرب من المحطة وقطع مع مقص. تجنب ثقب التجويف البريتوني. خفض في الساق لفضح الساق والفخذ و. قطع الأنسجة العضلية من عظام الساق باستخدام المقص. </li><li style=";text-align:right;direction:rtl"> لإزالة عظم الفخذ ، وقطع مرة واحدة اسفل الركبة ومرة ​​واحدة بالقرب من مفصل الورك. مكان العظام في طبق بتري الجرثومية التي تحتوي الباردة 1X PBS. لا تستخدم زراعة الأنسجة المعالجة أطباق بتري لهذه الخطوات ، لأن خلايا نخاع العظم ستلتزم بشكل دائم. استخدام مشرط لكشط قبالة اضافية الأنسجة العضلية. قطع العظم فوق الركبة بقليل لفضح نخاع. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من (ب) و (ج) لجمع نخاع العظم من عظم الفخذ الثاني. </li><li style=";text-align:right;direction:rtl"> استخدام حقنة مليئة 10cc 1X PBS الباردة وإبرة قياس 25 لطرد لنخاع العظم في أنبوب مخروطي 50 مل. يجب أن تتم إزالة العظام تظهر مرة واحدة بيضاء نقية نخاع. </li><li style=";text-align:right;direction:rtl"> تمرير الخليط PBS / نقي من خلال إبرة قياس 22 إلى تفتيت الحصى أي نخاع العظام. </li><li style=";text-align:right;direction:rtl"> تدور الخليط في 425 x ج لمدة 10 دقائق ، وإزالة طاف. </li><li style=";text-align:right;direction:rtl"> Resuspend الكرية في 8 مل من المتوسط ​​BMC. إضافة 1 مل من التعليق الخلية و 9 مل المتوسطة BMC إلى 8 أطباق بتري الجرثومية. </li><li style=";text-align:right;direction:rtl"> احتضان عند 37 درجة مئوية ، 5 ٪ CO 2. </li><li style=";text-align:right;direction:rtl"> في يوم 5 ، وإضافة 10 مل BMC المتوسطة على كل صفيحة. قبل يوم 7 ، وسوف تكون خلايا كاملة النمو. قد يكون passaged خلايا نخاع العظم لمدة 1-2 أسابيع بعد النضج. </li><li style=";text-align:right;direction:rtl"> لتقسيم خلايا نخاع العظم ، وإزالة المتوسطة وإضافة 5 مل من 1X PBS الباردة إلى كل طبق بتري. احتضان عند 4 درجة مئوية لمدة 30 دقائق ، حتى تبدأ الخلايا لرفع. خلايا نخاع العظم شطف قبالة مع لوحة ماصة نقل معقمة. </li><li style=";text-align:right;direction:rtl"> تجمع خلايا نخاع العظم في أنبوب مخروطي 50 مل وبيليه في 425 x ج لمدة 10 دقائق. </li><li style=";text-align:right;direction:rtl"> Resuspend وبيليه في 10 مل والمتوسطة BMC الاعتماد على عدادة الكريات. خلايا نخاع العظم في البذور 2x105 جيدا في صفيحة ال 24 جيدا مع الزجاج coverslips جولة في القاع. السماح للخلايا نخاع العظم قبل الانضمام بين عشية وضحاها مع اصابة T. يمكن reseeded الغوندية. خلايا نخاع العظام الزائدة على أطباق بتري لاستخدامها لاحقا. </li></ol> 4. اصابة الخلايا T. الغوندية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إصابة 25 سم 2 قارورة من HFFs متموجة مع T. 6 2x10 الغوندية وتنمو حتى تبدأ الخلايا ليز (حوالي 2-3 أيام). </li><li style=";text-align:right;direction:rtl"> استخدام مكشطة خلية لإزالة الخلايا المضيفة المصابة أحادي الطبقة من النسيج قارورة الثقافة ثم حرر الطفيليات من الخلايا المضيفة عن طريق تمرير المونولاير طردت من خلال إبرة عيار 27. </li><li style=";text-align:right;direction:rtl"> عدادة الكريات تستخدم لتحديد عدد الطفيليات في المتوسط. </li><li style=";text-align:right;direction:rtl"> إصابة 10 5 الطفيليات لكل بئر مع ساترة من monolayers HFF من البروتوكول 1 ، أو خلايا نخاع العظم من البروتوكول (3). السماح للغزو الطفيليات لمدة 3 ساعات في 37 درجة مئوية ، 5 ٪ CO 2. </li></ol> 5. بدء T. الغوندية المتباطئة التنمية من خلال الضغوط البيئية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> المتباطئة التنمية في HFFs مع زيادة درجة الحموضة والمتوسطة والتجويع CO 2 <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعد التنمية المتوسطة. التنمية المتوسطة يحتوي RPMأنا بدون بيكربونات 1640 ، 1 ٪ FBS ، 1 ٪ البنسلين الستربتوميسين ، و 42 HEPES ملم. الرقم الهيدروجيني إلى 8.0 تعقيم وتصفية. </li><li style=";text-align:right;direction:rtl"> إزالة DMEM HFFs من المصابين ، وشطف مع 1X PBS ، وإضافة 1ml متوسطة التنمية. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 3 أيام عند 37 درجة مئوية مع الهواء المحيط. </li></ol></li><li style=";text-align:right;direction:rtl"> تنشيط خلايا نخاع العظم <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد المتوسطة لتفعيل خلايا نخاع العظم. التنشيط هو BMC المتوسطة المتوسطة تستكمل مع الإنترفيرون γ 100U/ml وLPS نانوغرام / 100 مل. LPS يصوتن في حمام الماء لمدة 2 دقيقة قبل استخدامها لتعطيل المجاميع. </li><li style=";text-align:right;direction:rtl"> إزالة BMC المتوسطة من الآبار واستبدال مع 1 مل من المتوسط ​​التنشيط. </li><li style=";text-align:right;direction:rtl"> احتضان عند 37 درجة مئوية ، 5 ٪ CO 2 لمدة 3 أيام. </li></ol></li></ol> 6. الكشف المناعي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شطف المصابة الآبار ثلاث مرات مع 1X PBS. </li><li style=";text-align:right;direction:rtl"> إصلاح أحادي الطبقة مع 200ul من الفورمالديهايد 3 ٪ لمدة 20 دقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة تثبيتي ، وشطف الآبار مع 1X PBS. </li><li style=";text-align:right;direction:rtl"> إضافة 200μl من جليكاين M 0.1 إلى الآبار واسمحوا الجلوس لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> جليكاين إزالة الآبار وشطف مع 1X PBS. </li><li style=";text-align:right;direction:rtl"> إضافة 250 ميكرولتر من 3 ٪ BSA/0.2 ٪ TritonX - 100 في برنامج تلفزيوني ل1X permeabilize وعرقلة أحادي الطبقة لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> الحل إزالة الحجب وشطف الآبار مع 1X PBS. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر للتخفيف من 1:250 biflorus رودامين راصة Dolichos في 3 ٪ BSA/0.2 ٪ تريتون X - 100 في برنامج تلفزيوني 1X. </li><li style=";text-align:right;direction:rtl"> تغطية صحن ومكان على منصة شاكر في درجة حرارة الغرفة لمدة 1 ساعة </li><li style=";text-align:right;direction:rtl"> غسل coverslips 3 مرات مع 0.2 ٪ تريتون X - 100 في برنامج تلفزيوني 1X لمدة 5 دقائق على كل منصة شاكر. </li><li style=";text-align:right;direction:rtl"> coverslips جبل باستخدام VectaShield المتوسطة المتصاعدة التي تحتوي على 4&#39;6 - diamidino - 2 - phenylindole (دابي). </li><li style=";text-align:right;direction:rtl"> ملطخة T. ويمكن تصور الغوندية باستخدام المجهر الفلورسنت مع مناسبة لتصفية رودامين في التكبير من 100X. </li></ol> 7. ممثل النتائج الشكل 1 يوضح ممثل تلطيخ DBA ت. الغوندية BMMs في المنشط وHFFs تحت الضغط درجة الحموضة. كلا تلطيخ DBA تظهر حول الطفيليات التي تحتوي على فجوات ، مشيرا الى وجود مكونات جدار الكيس. صورة BMM تنشيط يبين تلطيخ DBA التي تنسجم مع السطح من فجوة. المقطع العرضي للT. الغوندية في درجة الحموضة وشدد HFFs يظهر الجدار كيسة مع عدم وجود الهياكل الداخلية الملون. <img src="/files/ftp_upload/2091/2091fig1.jpg" alt="الشكل 1" /> الشكل 1. ديسيبل تلون T. شدد وشدد الغوندية. الطفيليات بين الخلايا في ظل ظروف الإجهاد ، BMMs المنشط أو الحموضة HFFs ، كانت ملطخة DBA مترافق رودامين (الحمراء). الفرق المقابل تدخل (DIC) يبين الخطوط العريضة للكيسة وشريط أسود على نطاق يساوي 2 ميكرون. تجارب على الحيوانات. وقد أجريت تجارب على الحيوانات وفقا للمبادئ التوجيهية واللوائح التي وضعتها جامعة ويسكونسن ورعاية الحيوان اللجنة الاستخدام.

<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>

الإعلان عن أي تضارب في المصالح.

في حين ليست آلية التنمية المتباطئة مفهومة تماما ، والتحليلات الجينية الجزيئية ت. وقد أدى الغوندية مرحلة التحويل في مجال زراعة الأنسجة إلى اكتشاف الجينات التي تشارك في تشكيل كيسة المتباطئة 2،3،4. التحليلات أدى أيضا إلى ملاحظة أن يتم التعبير عن بعض علام?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<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

المقوسة الغوندية هو طفيلي تلزم بين الخلايا التي يمكن أن تغزو أي خلية الأنوية من الحيوانات من ذوات الدم الحار. خلال العدوى ، T. كما تنشر الغوندية شكل سريع تكرار يسمى tachyzoite. Tachyzoites تحويلها الى شكل بطيء النمو المتباطئة متكيس يسمى بعملية الإشارات التي لا تتميز بشكل جيد. داخل الحيوانات ، تم العثور على أكياس المتباطئة في النظام العصبي المركزي والأنسجة العضلية وتمثل المرحلة المزمنة من المرض. يمكن تحويلها إلى bradyzoites تكون محاكاة في مجال زراعة الأنسجة عن طريق التجويع CO 2 ، وذلك باستخدام المتوسطة مع الحموضة عالية ، أو إضافة انترفيرون غاما (IFNγ). تتميز Bradyzoites عن وجود جدار الكيس ، والتي lectin Dolichos biflorus راصة (DBA) بربط. يستخدم DBA fluorescently المسمى لتصور جدار الكيس في الطفيليات التي تزرع في الخلايا الليفية الإنسان القلفة (HFFs) التي تعرضت لانخفاض CO 2 ، وارتفاع متوسط ​​درجة الحموضة. وبالمثل ، والطفيليات المقيمين في نخاع العظام المستمدة من الفئران الضامة (BMMs) عرض الحائط كيسة كشفها بواسطة DBA بعد تنشيط BMMs مع IFNγ وlipopolysaccharide (LPS). وهذا البروتوكول لشرح كيفية إحداث تحويل T. الغوندية لbradyzoites باستخدام الرقم الهيدروجيني نمو عالية متوسطة منخفضة مع CO 2 وتفعيل BMMs. وسيتم تربيتها في الخلايا المضيفة coverslips ، tachyzoites المصابين هم من يمكنهم إما مع اضافة IFNγ وLPS (BMMs) أو تعرضها لدرجة الحموضة المتوسطة النمو العالية (HFFs) لمدة ثلاثة أيام. عند الانتهاء من العدوى ، وسوف تكون ثابتة الخلايا المضيفة ، permeabilized ، وحظرها. وسيتم استخدام الجدران كيسة تصور DBA رودامين مع المجهري مضان.

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

المقوسة الغوندية يحول الى شكل كيس ردا على الضغوط البيئية ، والتي يمكن في نماذج تحاكي زراعة الأنسجة. هذا الفيديو يوضح التقنيات لدراسة تشكيل جدار الكيس عن طريق تنشيط نخاع العظام المستمدة الضامة أو تغيير درجة الحموضة المتوسطة النمو في الخلايا الليفية.

التوكسوبلازما جدار الكيس الغوندية تشكيل المنشط تقوم البلاعم في نخاع العظام والمستمدة من الشروط المتباطئة

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. المقوسة الغوندية يحول الى شكل كيس ردا على الضغوط البيئية ، والتي يمكن في نماذج تحاكي زراعة الأنسجة. هذا الفيديو يوضح التقنيات لدراسة تشكيل جدار الكيس عن طريق تنشيط نخاع العظام المستمدة الضامة أو تغيير درجة الحموضة المتوسطة النمو في الخلايا الليفية.

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

الحصول على عالي النقاء المقوسة الغوندية البيضات من قبل التدرج كلوريد السيزيوم متقطع

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.

شاشة الوراثية لعزل التوكسوبلازما جوندياي المسوخ الخروج المضيف الخلية

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.

التحقيق في الاستقطاب البلاعم عن طريق نخاع العظم الضامة المشتقة

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.

التلاعب الجيني في<em&gt; Δku80</em&gt; سلالات لتحليل الجينوم الوظيفي لل<em&gt; التوكسوبلازما جوندياي</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.

نخاع العظام المستمدة من بلعم الإنتاج

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).

عزل وحقن في الوريد من الفئران نخاع العظام المستمدة حيدات

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.

الى الامام علم الوراثة شاشات طريق الضامة لتحديد<em&gt; التوكسوبلازما</em&gt; الجينات هامة الحصانة الذاتية للمقاومة إلى الإنترفيرون γ التي تعتمد على خلية

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.

توصيف الأيض من الاستقطاب M1 M2 الضامة والعظام نخاع المستمدة من طريق في الوقت الحقيقي التحليل خارج الخلية الجريان

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.

الكشف عن التنشيط إينفلاماسومي وموت الخلايا بيروبتوتيك في مورين الضامة المشتقة من نخاع العظام

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.

عزل البلاعم المشتقة من نخاع العظم واستزراعها في المختبر لدراسة بيولوجيا عدم الأكسدة والاختزال

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