بدعم من المنح المقدمة من المعاهد الوطنية للصحة (CA133085 إلى كوالا لمبور) والجمعية الأميركية للسرطان (RSG - 09 - 209 - 01 - TBG إلى كوالا لمبور).

1. التجريبية النموذجية الإنبثاث ماوس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في اليوم قبل حقن الخلايا السرطانية ، والبذور one T75 قارورة مع ما يصل إلى 1 × 10 7 CMS4 التقوا الخلايا في 10 مل من المتوسط ​​RPMI تحتوي على 10 ٪ للحصول على المصل سريعة النمو الخلايا السرطانية. يحضن بين عشية وضحاها في 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> في يوم من الحقن ، وإزالة المتوسطة وشطف خلايا مرة واحدة مع برنامج تلفزيوني ، ثم حصاد الخلايا السرطانية مع 0.05 ٪ التربسين - EDTA عند 37 لمدة 5 دقائق درجة مئوية. وقف تفاعل مع 10 مل من المتوسط ​​RPMI يحتوي المصل 10 ٪. نقل الخلايا إلى أنبوب مخروطي الشكل. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار في 1300 دورة في الدقيقة الخلايا في جهاز للطرد المركزي Sorvall RT الأيقونات لمدة 3 دقائق في درجة حرارة الغرفة. إزالة طاف. Resuspend الخلايا في HBSS 10 مل 1X جديدة ليغسل ، وتدور ثم تراجع مرة اخرى وresuspend بالطريقة نفسها. </li><li style=";text-align:right;direction:rtl"> عد الخلايا السرطانية على عدادة الكريات. تمييع الخلايا في HBSS ، بحيث يتم معلق الخلايا المطلوبة لكل الحقن في حجم ما مجموعه 100 ميكرولتر. </li><li style=";text-align:right;direction:rtl"> تدفئة 6-8 الاسبوع القديمة BALB / ج الفئران في كوب مغمورة في الماء الدافئ لتمدد الأوردة الذيل. ضع الماوس على ذيل restrainer الوريد على مقاعد البدلاء. استخدام microsyringe لحقن 100 ميكرولتر من الخلايا السرطانية في الوريد الذيل الأفقي. استخدام تقنية معقمة لتجنب العدوى. بعد الحقن ، وممارسة ضغوط طفيفة في موقع الحقن حتى توقف النزيف. </li><li style=";text-align:right;direction:rtl"> عودة الماوس إلى القفص ، والسماح للورم أن ينمو الى مرحلة المطلوبة. </li></ol> 2. السامة للخلايا الليمفاوية T - (CTL) نقل المناعي بالتبني <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في يوم من النقل ، ماصة صعودا وهبوطا لتنقية resuspend السامة للخلايا الليمفاوية تي ، أو CTLs ، أعد كما هو موضح في النص. نقل كافة الخلايا من واحد لوحة أنبوب مخروطي 15 مل ، عدم السماح للحجم تتجاوز أكثر من 2 / 3 من الأنبوب. </li><li style=";text-align:right;direction:rtl"> إدراج ماصة معقمة باستور في أنبوب مخروطي الشكل ، وتضع اللمفاويات متوسطة الفاصل ، أو LSM ، تحت الخلايا حتى تقترب من إجمالي حجم 14 مل. </li><li style=";text-align:right;direction:rtl"> يجب الحرص على عدم تعكير صفو طبقات. الطرد المركزي في 2000 دورة في الدقيقة لمدة 20 دقيقة في درجة حرارة الغرفة. لا تستخدم الفرامل لابطاء الدوار بعد الغزل. </li><li style=";text-align:right;direction:rtl"> نقل CTLs لأنبوب جديد 15 مل المخروطية. إضافة إلى HBSS وبلغ حجم التداول نحو 10 مل لغسل. </li><li style=";text-align:right;direction:rtl"> عدد الخلايا. تدور باستمرار في 1300 دورة في الدقيقة لمدة 5 دقائق في درجة حرارة الغرفة. Resuspend في HBSS لكثافة الخلايا المطلوبة لعمليات الحقن ، والحفاظ على الحجم الكلي للحقن في 100 ميليلتر. </li><li style=";text-align:right;direction:rtl"> استخدام حقن ذيل نفس الأسلوب كما رأينا في وقت سابق من هذا الفيديو لحقن CTLs في الفئران الحاملة للورم. </li><li style=";text-align:right;direction:rtl"> عودة الماوس إلى القفص ، والسماح للCTLs للتفاعل مع الورم. وعادة ما يتم التضحية الفئران لتحليل 14-21 يوما بعد العلاج CTL. </li></ol> 3. تصور الانبثاث الرئة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتصور الانبثاث الرئة ، ووضع الفأرة على ظهرها التضحية على لوحة الستايروفوم. دبوس الساقين لضمان الوصول دون عائق الى القصبة الهوائية. رش الجانب البطني مع الايثانول 70 ٪. </li><li style=";text-align:right;direction:rtl"> بدءا من منتصف البطن ، واستخدام المقص لقطع على طول خط الوسط ، من خلال القفص الصدري ، وحتى تجاه الغدد اللعابية. فضح القصبة الهوائية. استخدام الملقط لإزالة الأنسجة المحيطة القصبة الهوائية. </li><li style=";text-align:right;direction:rtl"> الصفحات 200 غيض ميكرولتر ماصة أسفل القصبة الهوائية. عقد قمة مع يد واحدة ، ورفع بلطف صعودا والقصبة الهوائية بعيدا عن الجسم. </li><li style=";text-align:right;direction:rtl"> منصة تدوير 180 درجة. استخدام الحقنة 50 مل لحقن الحبر الهند إلى الرئتين عبر القصبة الهوائية. تضخم الرئتين تماما مع الحبر حتى تشعر أنك على مقاومة قوية. </li><li style=";text-align:right;direction:rtl"> استخدام مقص لقطع القصبة الهوائية. زوج من الشرائح ملقط تحت الرئتين وانتشالهم من الفأرة. شطف الرئتين لفترة وجيزة في كوب من الماء 1L. </li><li style=";text-align:right;direction:rtl"> في غطاء الدخان الكيميائية ، ونقل إلى الرئتين قارورة زجاجية تحتوي على 5 التلألؤ مل من محلول Fekete ل. سوف تظهر وكأنها نسيج الورم عقيدات بيضاء على الرئتين سوداء بعد بضع دقائق. ويمكن الآن للأورام تحسب وتخزينها في حل Fekete الى أجل غير مسمى. </li></ol> 4. ممثل الانبثاث في الرئتين متصور <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> هنا ، والرئتين من الفئران التي حقنت مع سرطان الثدي 4T1 خط الخلية تظهر بقع بيضاء تشير إلى أورام. الفئران التي حقنت بخلايا 4T1 transfected مع K79E المهيمنة سلبية IRF8 متحولة تبرز إمكانات تعزيز المنتشر من الخلايا السرطانية. </li><li style=";text-align:right;direction:rtl"> هذه الصور تظهر التحليل النسيجي للفعالية العلاج المناعي CTL نقل بالتبني. وأظهرت الفئران الحاملة للورم حقنها بمحلول ملحي أورام متعددة 6 أيام في وقت لاحق ، أشارت بواسطة الأسهم الصفراء (A). فئران حقنت بخلايا T ورم محددة ، من ناحية أخرى ، أظهرت انخفاضا من الأورام (B). </li><li style=";text-align:right;direction:rtl"> في التجربة نفسها ، الهند علاج الحبر يعطي أبسط طريقة لقياس مدى فعالية نقل بالتبني CTL. هنا ، عقيدات بيضاء على الورم الحاملة للورم الرئة التمييز بوضوح بينها وبين الرئتين التي مرت بنجاح نقل بالتبني CTL ، والعد العقيدات يسمح للحصول على درجةمن الكميات غير ممكن مع الأنسجة. </li></ol> 5. ممثل النتائج <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="الشكل 1" /> الشكل 1. مخطط تجريبي لالانبثاث ورم التجريبية وCTL المناعي نقل التكيف نموذج الفأر. النقاط الحمراء تشير إلى الخلايا السرطانية والنقاط الخضراء تشير CTLs. <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="الشكل 2" /> تعزيز الشكل 2. IRF8 تعطيل وظيفة المحتملة المتنقل من الخلايا السرطانية. وكان transfected ستابلي الماوس الخلية 4T1 سرطان الثدي مع خط ناقل (4T1.Vector) أو ناقلات إبداء IRF8 المهيمنة سلبية متحولة K79E (4T1.IRF8K79E) (15 ، 16). تم حقن الخلايا السرطانية الرابع في ذيل الفأر الأوردة الجانبية. فقد ضخمت الورم الحاملة الرئتين مع الحبر الهند لتصور العقيدات الورم. وينظر بسهولة العقيدات الورم على شكل بقع بيضاء على خلفية سوداء نسيج الرئة. <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="الشكل 3" /> الشكل 3. تحليل النسيجي من فعالية العلاج المناعي CTL نقل بالتبني. تم حقن الخلايا ساركومة خط الماوس CMS4 التقوا الرابع في أوردة الذيل الماوس الجانبية. بعد ثلاثة أيام ، تم حقن المياه المالحة (A) أو ورم محددة الخلايا التائية (B) في الفئران الحاملة للورم. وقد تم تحليل الرئتين بعد ستة أيام من العلاج بواسطة CTL تلطيخ H &amp; E التقليدية النسيجية. يشار العقيدات الورم عن طريق الأسهم. <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="الشكل 4" /> الشكل 4. التصور العقيدات الورم عن طريق التضخم الحبر الهند. تم حقن الخلايا ساركومة خط الماوس CMS4 التقوا الرابع في أوردة الذيل الماوس الجانبية. بعد ثلاثة أيام ، تم حقن المياه المالحة (مراقبة) أو ورم محددة الخلايا التائية (+ CTL) في الفئران الحاملة للورم. وقد تم تحليل الرئتين أربعة عشر يوما بعد العلاج CTL. بقع بيضاء ، والتي هي العقيدات الورم ، والسماح للالكمي سهل من فعالية العلاج CTL.

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تم شراؤها من الفئران في معهد السرطان الوطني (Friderick ، ​​MD) ، ومقرها في كلية الطب في جورجيا مرفق الحيوانية. وكانت التجارب والرعاية / الرعاية بالاتفاق مع الأنظمة الاتحادية وبروتوكولا وافق عليها كلية الطب في جورجيا IACUC اللجنة.

الحلول : الهند محلول الحبر (17) : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> صب 150 مل من الماء المقطر إلى 250 مل من القارورة. </li><li style=";text-align:right;direction:rtl"> 4 إضافة هيدروكسيد الأمونيوم إلى قطرات الماء المقطر. </li><li style=";text-align:right;direction:rtl"> إضافة 30 مل الحبر الهند الأسهم (أي سانفورد السحر الاسود ماء الحبر رسم 4465 البند 44011) إلى خليط النشادر والماء. </li><li style=";text-align:right;direction:rtl"> أعلى قبالة مع الماء المقطر لحجم 200 مل. الحل هو على استعداد للحقن. </li></ol> Fekete محلول (17) : ويستخدم حل Fekete للهند تبييض الحبر تضخم الورم الحاملة الرئتين لتمييز الورم العقيدات بيضاء من خلفية سوداء من الأنسجة الطبيعية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 350ml EtOH 95 ٪ ل1L زجاجة. </li><li style=";text-align:right;direction:rtl"> إضافة الماء المقطر 150ml </li><li style=";text-align:right;direction:rtl"> إضافة 50ml الفورمالديهايد </li><li style=";text-align:right;direction:rtl"> إضافة حمض 25ml الحمضية الجليدية </li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

الفأر نموذج تجريبي الانبثاث الانبثاث هو نموذج بسيط ولكن ذات الصلة من الناحية الفسيولوجية. يتم حقن الخلايا السرطانية عن طريق الوريد (رابعا) في أوردة الذيل الماوس واستعمار في الرئتين ، وبالتالي ، تشبه الخطوات الأخيرة من خلايا الورم ورم خبيث عفوية : البقاء في التسرب وتداولها والاستعمار في الأجهزة الطرفية. من وجهة نظر علاجية ، والنموذج التجريبي الانبثاث هو النموذج المثالي منذ أبسط والهدف من العلاجات غالبا ما تكون نقطة النهاية في الانبثاث : الورم النقيلي أنشئت في الجهاز البعيد. في هذا النموذج ، يتم حقن الخلايا السرطانية في أوردة الذيل الرابع الماوس ويسمح للاستعمار وتنمو في الرئتين. ثم يتم حقن الورم محددة CTLs الرابع في الماوس الانبثاث الحاملة. يمكن التحكم في عدد وحجم الرئة الانبثاث من قبل عدد من خلايا الورم ليتم حقنه والوقت للنمو الورم. لذلك ، مراحل مختلفة من ورم خبيث ، من الحد الأدنى لالانبثاث الانبثاث واسعة ، يمكن أن تكون على غرار. ويتم تحليل الانبثاث الرئة بسبب التضخم مع الحبر ، مما يتيح سهولة الملاحظة البصرية والكمي.

Watch this Scientific Journal Video about Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model at JoVE.com

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

والانبثاث الرئة التجريبية وCTL نموذج الفأر المناعي لتحليل التفاعل خلية ورم الخلايا التائية<em&gt; في الجسم الحي</em&gt;.

الإنبثاث التجريبية وCTL المناعي نقل بالتبني النموذجي ماوس

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

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JoVE Journal

Immunology and Infection

19.3K Views.  Medical College of Georgia. والانبثاث الرئة التجريبية وCTL نموذج الفأر المناعي لتحليل التفاعل خلية ورم الخلايا التائية في الجسم الحي.

Video: Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

بروتوكولات لتلقيح المهبل وجمع العينات في النموذج التجريبي للماوس المبيضات المهبل

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

التغلب على عدم التجاوب في التجارب الذاتية التهاب الدماغ (EAE) سلالات الفئران المقاومة للنقل بواسطة بالتبني والتحدي الأنتيجين

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

المعجل نوع 1 الحث السكري في الفئران عن طريق نقل بالتبني من CD4 + T خلايا سكري

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

العزلة وتنقية وصفها من العظام بالنسبة للفئران العدلات نخاع للدراسات الفنية والتجارب نقل بالتبني

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

الخلايا الجذعية داخل الوعاء اللمفي والتطعيم في الفئران

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

تقييم تطوير خلايا الفئران الجذعية بلازماوية في بقع باير طريق نقل بالتبني من أسلاف المكونة للدم

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

جيل من الخلايا T CAR للعلاج بالتبني في سياق ورم أرومي دبقي مستوى الرعاية

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

تحريض الفئران المعوية التهاب عن طريق نقل بالتبني من المستجيب CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; عالية</sup&gt; خلايا T في الفئران العوز المناعي

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

نقل الخلايا المناعية السامة للخلايا في نماذج مورين لتكثيفها عديدة الأشكال جليوبلاستوما أورثوتوبيك الإنسان التبني المناظير باستخدام الأشعة

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

إنشاء نموذج الماوس ثنائي الأنسنة TK-NOG لمسببات أمراض الكبد المرتبطة بفيروس نقص المناعة البشرية

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...