وأيد هذا العمل من قبل منظمة العفو الدولية NIH P01 منح 068730 (SNC ، NS) ، ومنح CA016520 المعاهد الوطنية للصحة / TAPITMAT (NS) ، والتمويل من القطاع الخاص من مؤسسة Claneil (NS) ، والمبيض بوغ الإطارية بشأن تغير المناخ ، ومنحة لجامعة ولاية بنسلفانيا ( CA83638 P50) وفوكس تشيس للسرطان مركز الأساسية غرانت (P30 CA06927) (DCC). المؤلفان بالشكر إلى المساعدة التقنية الممتازة لمرفق كور البصرية / ضوء بارد من إخراج الدكتور EJ Delikatny في جامعة بنسلفانيا انتوني Secreto ، من الخلايا الجذعية الأساسية طعم أجنبي وإخراج G. Desnoyers - دانيه الدكتور في جامعة بنسلفانيا للسرطان مركز للتدريب لSNC تقنية الحقن مثلي وDangaj Denada في جامعة بنسلفانيا / OCRC للمساعدة في عملية جراحية.

1. خلية ثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل الحقن مثلي ، والثقافة MOV1 خلايا كات ، والمستمدة من DR26 الأورام ، في قارورة T175 حتى انهم 90 ٪ متموجة. خطة لاستخدام 1-5000000 الخلايا في الحقن ، وهو ما سيتطلب 1 أو 2 T175 القوارير. </li><li style=";text-align:right;direction:rtl"> في يوم من الحقن ، وحصاد الخلايا وتحديد عدد الخلايا باستخدام عدادة الكريات. </li><li style=";text-align:right;direction:rtl"> مرة واحدة وقد تم تحديد تركيز الخلية ، بيليه الخلايا بواسطة الطرد المركزي لمدة 5 دقائق في ز X 300 في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> بعد زيادة ونقصان ، resuspend الخلايا لديك 1 مليون نسمة في 10 microliters من برنامج تلفزيوني العقيمة مع EDTA 0.002 M </li></ol> 2. قبل الجراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل الجراحة ، وتملأ حقنة الأنسولين مع الخلايا 3/10cc MOV1 كات - 1 مليون في 10 microliters EDTA من برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> نقل ماوس isofluorane تخدير لوحة التدفئة. إضافة مرهم العين لمنع جفاف العين. ثم ، تضاف فورا رأس الحيوان في مخروط الأنف نظام متصلة المرذاذ isoflurane لتسليم جميع أنحاء التخدير الجراحة. </li><li style=";text-align:right;direction:rtl"> بعد تطهير مكان الحقن مع مسحات الكحول ، وحقن تحت الجلد 5 ملغ / كلغ من كيتوبروفين ، وهو مسكن ما قبل الجراحة ، مع حقنة الانسولين 3/10cc. </li><li style=";text-align:right;direction:rtl"> باستخدام مقص ، حلاقة الجزء الأيسر من الذيلية ظهر من مفرق الصدرية الى قاعدة الذيل للحيوانات. تطبيق كريم إزالة الشعر لإزالة الشعر. ثم ، إزالة الزائدة مع منشفة ورقية مبللة. </li><li style=";text-align:right;direction:rtl"> بمجرد إزالة الشعر ، وتعقيم المنطقة حلق مع بوفيدون اليود ومسحات الكحول. ثم ، وضع ثنى الجراحية في جميع أنحاء المنطقة من شق. </li></ol> 3. جراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل الجراحة ، وضبط مستويات المرذاذ isoflurane إلى 1.5 ٪. </li><li style=";text-align:right;direction:rtl"> تحقق من أن هذا الحيوان هو تخدير تماما معسر لوحة القدم. </li><li style=";text-align:right;direction:rtl"> المقبل ، وحدد موقع الطحال تحت الجلد. ثم استخدام مقص جراحي ، وجعل شق ظهراني 1-2 سم طويلة على أعلى يمين الطحال. </li><li style=";text-align:right;direction:rtl"> تشريح الحيز خلف الصفاق. وسيراعى في لوحة المحيطة المبيض الماوس. استخدام ملقط منحنية لفهم وفضح لوحة الدهون المحيطة المبيض الماوس. </li><li style=";text-align:right;direction:rtl"> هيدرات الجهاز مع بعض قطرات من برنامج تلفزيوني العقيمة. </li><li style=";text-align:right;direction:rtl"> استخدام ملقط منحنية لفهم ، يتراجع... موقف... ومن ثم تأمين المبيض للحقن </li><li style=";text-align:right;direction:rtl"> بينما يمسك بحزم المبيض مع ملقط ، وضخ 10 microliters MOV1 من الخلايا السرطانية في المبيض كات. وهناك إدراك الشركة منع تسرب السوائل أو قلس. </li><li style=";text-align:right;direction:rtl"> مباشرة بعد الحقن ، والافراج عن التوتر التي تبذلها ملقط. ينبغي أن ثقب في المبيض يتراجع عفويا وثيق. </li><li style=";text-align:right;direction:rtl"> باستخدام حمض خياطة Polyglycolic امتصاص المرفقة إبرة ، أغلق الجرح الرجعية البريتوني. </li><li style=";text-align:right;direction:rtl"> الافراج عن الحيوان من مخروط الأنف. </li><li style=";text-align:right;direction:rtl"> تمدد الجلد وختم حواف الجرح ظهراني مع بضع قطرات من لاصق الأنسجة. </li><li style=";text-align:right;direction:rtl"> أخيرا ، تدير 100 microliters شفويا من المضادات الحيوية للحيوانات. ثم ضع مرة أخرى في قفص ، ورصد لتحقيق الانتعاش. ابقاء الحيوانات على المضادات الحيوية في مياه الشرب لمدة أسبوع. </li></ol> 4. التصوير في فيفو <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد أسبوع واحد مثلي من حقن الخلايا السرطانية MOV1 كات ، نفذ في التصوير الجسم الحي. تبدأ عن طريق نقل ماوس isofluorane تخدير إلى غرفة التصوير. </li><li style=";text-align:right;direction:rtl"> بدوره مستوى المرذاذ isoflurane وصولا الى 2 ٪. </li><li style=";text-align:right;direction:rtl"> فيفو أداء في مجال التصوير وفقا لتعليمات الشركة المصنعة لنظام التصوير. في هذا الفيديو ، سيتم استخدام نظام لومينا. </li><li style=";text-align:right;direction:rtl"> لصورة ، انقر فوق البرنامج صورة حية رمز سطح المكتب. ثم في لوحة التحكم IVIS اقتناء ، حدد &quot;تهيئة&quot;. ضبط الصك هي مماثلة لإعدادات الكاميرا </li><li style=";text-align:right;direction:rtl"> على اقتناء لوحة التحكم إعداد المعلمات حيازة الصك. لمضان ، تحقق &quot;نيون&quot;. فوق مربع الصورة للحصول على صورة فوتوغرافية مع كل صورة. </li><li style=";text-align:right;direction:rtl"> المقبل ، ضبط الوقت والتعرض للسيارات. تحت عنوان &quot;binning بكسل أو قرار اتفاقية مكافحة التصحر&quot; ، تحقق &quot;متوسطة&quot;. ثم وكيل F / إيقاف أو الفتحة ، والتحقق من قيمة 2. المقبل ، حدد تصفية 535 الإثارة وتصفية DsRed الانبعاثات. </li><li style=";text-align:right;direction:rtl"> تحت مجال الرؤية ، انقر على الصورة لمشاهدة باء ماوس واحدة. </li><li style=";text-align:right;direction:rtl"> ثم انقر على اكتساب للبدء في الحصول على الصور. </li><li style=";text-align:right;direction:rtl"> بمجرد الحصول على الصور اكتمال استخدام منطقة سياحية ، أو العائد على الاستثمار ، وأداة لقياس الإشارة. انقر على أيقونة قياس للافراج عن القيم إشارة والمنطقة. </li><li style=";text-align:right;direction:rtl"> أخيرا ، انقر على &quot;حفظ&quot; لحفظ الصورة في مجلد المستخدم </li><li style=";text-align:right;direction:rtl"> بعد أن تم حفظ الصور ، والتوقف عن إقامة isoflurane والعودة إلى قفصه الماوس. يجب الماوس يستيقظ على الفور. </li></ol> 5. ممثل النتائج التي تستخدم هذا البروتوكولويمكن رصد النمو في الجسم الحي من سرطان المبيض مثلي ما لا يقل عن 3 أسابيع باستخدام إجراء غير الغازية. <img src="/files/ftp_upload/2146/2146fig1.jpg" alt="الشكل 1" /> تم حقن orthotopically الشكل 1. MOV1 خلايا كات ، أو برنامج تلفزيوني على السيطرة السلبية ، في المبيض من الفئران غير المعرضة للورم (حق الحيوان والحيوان واليسار على التوالي) ، وفي التصوير فيفو أجري 2 أسابيع في وقت لاحق. تم قياس الانبعاثات الناتجة عن مضان خلايا كات MOV1 engrafted في المبيض وذلك بالمقارنة مع من الفأر السيطرة السلبية. <img src="/files/ftp_upload/2146/2146fig2.jpg" alt="الشكل 2" /> وكانت ملطخة الشكل 2. أقسام مجمدة MOV1 من أورام المبيض كات مع CD4 مكافحة biotinylated خريطة موقع تليها الركيزة DAB (بني غامق) للكشف عن الاورام الليمفاوية التسلل (السهم الأسود). وcounterstained الخلايا مع الأخضر الميثيل لتصور نواة الخلية (الأزرق). وكانت الخلايا السرطانية (السهام الحمراء) شكليا متميزة من خلايا تي. الشريحة يظهر تضخيم 40X.

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الإعلان عن أي تضارب في المصالح.

الجراحة والحقن مثلي حقن مثلي في جراب المبيض مطالب التدريب والدقة. وبالتالي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في حالة سوء الممارسة الجراحية والخبرة مع الجثث الاولى. </li><li style=";text-align:right;direction:rtl"> استخدام الإناث تفضيل...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM-GLUTAMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10564-011</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14040</td>
<td>&nbsp;</td>
<tr>
<td>Versene</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-711E</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Deltaphase</td>
<td>39 DP</td>
<td>&nbsp;</td>
<tr>
<td>Povidone pads</td>
<td>&nbsp;</td>
<td>Dynarex</td>
<td>1108</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>06-669-62</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tears ointment </td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>17845-153</td>
<td>&nbsp;</td>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Fort Dodge laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3cc/insulin syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309301</td>
<td>&nbsp;</td>
<tr>
<td>Polyg Polyglycolic Acid suture/needle (3/8 19mm)</td>
<td>&nbsp;</td>
<td>Syneture</td>
<td>9612-31</td>
<td>&nbsp;</td>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>Vetbond</td>
<td>3M</td>
<td>&nbsp;</td>
<tr>
<td>Vet Bactrim/ oral suspension</td>
<td>&nbsp;</td>
<td>Hi-tech Pharmacal</td>
<td>840823</td>
<td>&nbsp;</td>
<tr>
<td>IVIS-Lumina</td>
<td>&nbsp;</td>
<td>Caliper lifesciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isofluorane</td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>J108013</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum, Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437036</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>TurboFP635 mammalian vector</td>
<td>&nbsp;</td>
<td>Evrogen</td>
<td>FP721</td>
<td>&nbsp;</td>
<tr>
<td>T175 flasks</td>
<td>&nbsp;</td>
<td>cellstar</td>
<td>660-190</td>
<td>&nbsp;</td>		</tbody>
		</table>
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an orthotopic model of serous ovarian cancer in immunocompetent mice for in vivo tumor imaging and monitoring of tumor immune responses

خلفية : عادة يتم تشخيص سرطان المبيض في مرحلة متقدمة حيث نسبة حالة / وفيات مرتفعة وهكذا يظل الأكثر فتكا من جميع سرطانات الجهاز التناسلي للمرأة بين النساء في الولايات المتحدة 1،2،3. الأورام المصلية هي الأشكال الأكثر انتشارا من سرطان المبيض وراثيا على 4،5 TG - MISIIR أبوغزاله يمثل نموذج الفأر الوحيد الذي يتطور بشكل عفوي هذا النوع من الأورام. TG - MISIIR أبوغزاله الفئران أعرب SV40 تحويل المنطقة الخاضعة لسيطرة العقاقير المثبطة مولر النوع الثاني من مستقبلات (MISIIR) مروج الجين 6. وقد تم تحديد خطوط إضافية المعدلة وراثيا التي تعبر عن العلامات SV40 والتحوير ، ولكن لا تطور أورام المبيض. ورم غير الفئران المعرضة للمعرض عمر نموذجية لC57Bl / 6 الفئران وخصبة. ويمكن استخدام هذه الفئران كمتلقين طعم خيفي مسانج للخلايا السرطانية المعزولة من TG - MISIIR - TAG - DR26 الفئران. الهدف : على الرغم من التصوير الورم ممكن 7 ، الكشف المبكر عن الأورام العميقة الصعبة في الحيوانات الحية الصغيرة. لتمكين الدراسات قبل السريرية في نموذج حيواني سليمة مناعيا لسرطان المبيض المصلي ، وصفنا نموذج الفأر مسانج لهذا النوع من سرطان المبيض الذي يسمح التصوير في الجسم الحي ، ودراسات للالمكروية الورم ورم والاستجابات المناعية. طرق : أولا نحن المستمدة TAG سرطان الماوس + خط الخلية (MOV1) من ورم المبيض العفوي المقطوع في الأسبوع 26 من العمر من العمر TG - MISIIR أبوغزاله DR26. ثم ، فإننا transduced ستابلي MOV1 الخلايا مع متجه الفيروسة البطيئة TurboFP635 الثدييات التي تكود Katushka ، متحولة بعيدة للبروتين أحمر فلوري في البحر الأحمر من Entacmaea quadricolor النعمان مع الإثارة / الانبعاثات في ماكسيما 588/635 8،9،10 نانومتر. نحن مزروع orthotopically MOV1 كات في المبيض 11،12،13،14 غير الورم الفئران TG - MISIIR أبوغزاله الإناث عرضة. وأعقب تطور الورم عن طريق التصوير الضوئي في الجسم الحي والمكروية الورم كان تحليلها من قبل المناعية. المتقدمة مزروع Orthotopically MOV1 خلايا أورام المبيض كات مصلي : النتائج. يمكن MOV1 الأورام كات يمكن تصور من قبل في مجال التصوير فيفو تصل إلى ثلاثة أسابيع بعد الزرع (الشكل 1) وكانت مخترقة مع الكريات البيض ، كما لوحظ في سرطان المبيض الإنسان 15 (الشكل 2). الاستنتاجات : نحن تصف نموذجا مثلي من سرطان المبيض مناسبة للتصوير في الجسم الحي من الأورام في وقت مبكر نظرا لارتفاع درجة الحموضة في المحافظة على استقرار وصمود للضوء من Katushka في الأنسجة العميقة. نقترح استخدام هذا النموذج مسانج رواية الاصابة بسرطان المبيض لمصلي في الدراسات المجراة التصوير ورصد الاستجابات المناعية الورم والمناعية.

Watch this Scientific Journal Video about An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses at JoVE.com

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

لدراسة<em&gt; في الجسم الحي</em&gt; نمو الورم ورم والمكروية ، استخدمنا نموذج الفأر مسانج ومثلي من سرطان المبيض في الحيوانات مناعيا. transduced نحن خلية الورم خط الماوس (MOV1) مع بروتين فلوري Katushka (MOV1<sup&gt; كات</sup&gt;) ، وهنا نعرض لها مثلي في زرع المبيض و<em&gt; في الجسم الحي</em&gt; التصوير.

نموذج مثلي من سرطان المبيض في الفئران مصلي مناعيا ل في الجسم الحي التصوير الورم ورصد الاستجابات المناعية ورم

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

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Research

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Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

18.1K Views.  University of Pennsylvania-School of Medicine. لدراسة في الجسم الحي نمو الورم ورم والمكروية ، استخدمنا نموذج الفأر مسانج ومثلي من سرطان المبيض في الحيوانات مناعيا. transduced نحن خلية الورم خط الماوس (MOV1) مع بروتين فلوري Katushka (MOV1 كات) ، وهنا نعرض لها مثلي في زرع المبيض و في الجسم الحي التصو?...

Video: An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

ل<em&gt; في المختبر</em&gt; نموذج لقياس الاستجابات المناعية لمكافحة الملاريا في سياق الفيروس المسبب للإيدز

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

و إيل-8 عابر نموذج الماوس المعدلة وراثيا ل<em&gt; في فيفو</em&gt; رصد طويل الأمد للردود الالتهابية

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

عزل السالمونيلا تيفيموريوم-التي تحتوي على فاجوسوميس من الضامة

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

الخياطة في فيفو الاختبارات الخلوية لدراسة المناعي في الأورام الخاصةCD8 + T الاستجابات الخلية

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

غرس ورصد من قبل PET/CT من نموذج المجسم من ورم الظهاره المخروطية البشرية الجنبي في الفئران Athymic

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

التصور، القياس الكمي، ورسم خرائط للخلايا المناعية في البيئة الدقيقة الورم

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

رسم خرائط دقيقة للدماغ لأداء المتكررة في التصوير فيفو من ديناميات العصبية المناعية في الفئران

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

نماذج الاستزراع المشترك للموائع الدقيقة لتشريح الاستجابة المناعية في البيئات الدقيقة للورم في المختبر

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

عدوى الإشريكية القولونية المتكررة في المسالك البولية الناجمة عن التعرض للمثانة المهبلية Gardnerella في الفئران

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.