وقدم الدعم من قبل تقنيات بيكتون ديكنسون. الآراء الواردة في هذا البيان هي آراء المؤلفين ولا تدعي لتعكس السياسة الرسمية للحكومة الولايات المتحدة. وقد أجريت بحوث في الامتثال لقانون رعاية الحيوان والقوانين الاتحادية واللوائح الأخرى المتعلقة الحيوانات والتجارب التي تنطوي على الحيوانات ويلتزم المبادئ المنصوص عليها في دليل لرعاية واستخدام الحيوانات المختبرية، المجلس القومي للبحوث، عام 1996. وتماما المعتمدين منشأة حيث تم إجراء هذا البحث من قبل جمعية تقويم والاعتماد من المنظمة الدولية لرعاية الحيوان المعملية.

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Immunol. 168, 986-990 (2002).</li><li>Rangel-Moreno, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CXC+chemokine+ligand+13,+CC+chemokine+ligand+(CCL)+19,+and+CCL21+in+the+organization+and+function+of+nasal-associated+lymphoid+tissue.">Role of CXC chemokine ligand 13, CC chemokine ligand (CCL) 19, and CCL21 in the organization and function of nasal-associated lymphoid tissue.</a> J. Immunol. 175, 4904-4913 (2005).</li><li>Morefield, G. L., Hawkins, L. D., Ishizaka, S. T., Kissner, T. L., Ulrich, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+Toll-like+receptor+4+agonist+enhances+vaccine+efficacy+in+an+experimental+model+of+toxic+shock+syndrome.">Synthetic Toll-like receptor 4 agonist enhances vaccine efficacy in an experimental model of toxic shock syndrome.</a> Clin. Vaccine Immunol. 14, 1499-1504 (2007).</li></ol>

الإعلان عن أي تضارب في المصالح.

قدمنا ​​وسائل جماعية لتطوير نموذج حيواني، والحصول على العينات البيولوجية، وفحوصات لدراسة NALT المرتبطة الاستجابات المناعية 1-4. هناك عوامل إضافية للنظر في أثناء أداء هذه الأساليب. وينبغي اتباع الأساليب العقيمة معيار للجراحة وزراعة الأنسجة. وهناك مجموعة من العوامل المضادة للبكتيريا ومضاد للفطريات التي استخدمت خلال العزلة والثقافة، فضلا عن الحفاظ على الآلات المعقمة، مجال العمل، والأذواق تطهيرها لحد من مخاطر التلوث. وينبغي أن يتم فحص اللعاب، ويغسل الأنف وعينات مماثلة إفراز المخاطية للتلوث محتمل مع الدم، كما تم العثور على أضداد المصل بشكل عام في تركيزات أعلى. كذلك، يجب فقط الافرازات المخاطية أن تضعف قليلا لتحليل لتركيزات أقل من الأجسام المضادة موجودة في هذه العينات بالمقارنة مع المصل. يجب أن تبقى الفئران دافئ مباشرة بعد عملية جراحية لمرحلة ما قبلتنفيس عن إمكانية التخدير التي يسببها انخفاض حرارة الجسم. الفئران بديل يستريح على جنوبهم أثناء النقاهة بعد الجراحة لتقليل تنفس غير منتظم. الإجراء الجراحي هو أكثر كفاءة مع ثلاثة أشخاص يعملون معا لإتمام هذه المهام: 1 أداء عملية جراحية، واحدة للمساعدة في عقد فتح الفم، واحد لتوفير الرعاية بعد العمليات الجراحية مثل الفئران على التعافي من التخدير. فمن الضروري استخدام H &amp; E تلطيخ من المقاطع عبر الجمجمة في نهاية كل الإجراءات التجريبية أو دراسات للتحقق من نجاح عملية جراحية لكل فأر. نتائج الجراحة ممكنة هي: الكامل والثنائية الاجتثاث NALT، الاجتثاث غير مكتملة، أو NALT سليمة. ويمكن استخدام ذلك لأن ليس كل العمليات الجراحية سوف يؤدي إلى فقدان كامل للNALT والحيوانات مع NALT المتبقية أو سليمة والضوابط الداخلية. آخر النتائج المحتملة هو أن الحنك فشل للشفاء تماما، وترك فتحة توصيل الأنف والتجاويف عن طريق الفم. غير كاملوالشفاء من الحنك يؤدي إلى انخفاض الوزن وعدم قدرته على النمو، ويجب إزالة هؤلاء الأفراد من الدراسات. وسوف تدرس الردود اللقاح الأول مع نموذج الفأر تعمل على إنشاء دور للNALT في نتائج الدراسة المقصود (استجابة الأجسام المضادة، والبقاء على قيد الحياة، وما إلى ذلك). جراحيا إزالة NALT يسهل تحديد الاشتراكات الأنف لحصانة المحلية والنظامية. النهج الجراحية وصفها هنا هو الأسلوب الأكثر مباشرة للحصول على نموذج الفأر تخلو من NALT. حدد بنظام خروج المغلوب وقد أبلغ عن نماذج الماوس إلى NALT نقص، ولكن هذه الحيوانات هي أيضا من نقص في السيتوكينات أو كيموكينات أساسي في تطوير الأنسجة الأخرى اللمفاوية الثانوية، ويمكن أن تؤوي عيوب إضافية 12،13. علاوة على ذلك، تم تطوير أساليب وصفها هنا لدراسة جوانب عديدة من الاستجابات المناعية تنشأ داخل الممرات الأنفية. وتستند النتائج التي حققناها في دراسات تجريبية باستخدام كامل الحنك العلويه من الماوس لزراعة الأنسجة، على الرغم من أنه من الممكن أن يمكن استخدام المقاطع. أخيرا، فإن النموذج NALT مثقف مفيد لإجراء التجارب تماما في زراعة الأنسجة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> الفولاذ المقاوم للصدأ العقيمة شفرات جراحية، رقم (11) </td><td> Miltex </td><td> 4-311 </td><td></td></tr><tr><td> مقبض السكين، رقم (3) </td><td> Miltex </td><td> 4-7 </td><td></td></tr><tr><td> 48-جيدا لوحات خلية الثقافة </td><td> Costar </td><td> 3548 </td><td></td></tr><tr><td> RPMI 1640 </td><td> إينفيتروجن </td><td> 11875-093 </td><td></td></tr><tr><td> الأبقار مصل الجنين، والحرارة المعطل </td><td> Gibco / إينفيتروجن </td><td> 16000-044 </td><td> اضرب النهائية: 10٪ من حيث الحجم في وسائل الإعلام ثقافة </td></tr><tr><td> ستربتومايسين سلفات </td><td> سيغما </td><td> S9137 </td><td> اضرب النهائية: 100 ميكروغرام / مل </td></tr><tr><td> بنسلين </td><td> سيغما </td><td> P7794 </td><td> اضرب النهائية: 100 UI / مل </td></tr><tr><td> جنتاميسين </td><td> سيغما الدريخ </td><td> G1397-10ML </td><td> اضرب النهائية: 50 ميكروغرام / مل </td></tr><tr><td> Fungizone </td><td> Gibco / إينفيتروجن </td><td> 15290-018 </td><td> اضرب النهائي: 1 ميكروغرام / مل </td></tr><tr><td> HEPES </td><td> سيغما الدريخ </td><td> H0887 </td><td> اضرب النهائية: 10 مم </td></tr><tr><td> أنابيب إيبندورف Microcentrifuge </td><td> إيبندورف </td><td> 022364111 </td><td></td></tr><tr><td> نوترا جل الكرز الغذاء الفأر بنكهة الرطب </td><td> الحيوي خدمة </td><td> S4798-TRAY </td><td></td></tr><tr><td> Metacam </td><td> بورنغير إنغلهايم </td><td> 601531000 </td><td> قطرة واحدة من التعليق مغ / 1.5 مل عن طريق الفم </td></tr><tr><td> الكيتامين </td><td> فايزر </td><td> 00856440301 </td><td> نهائي المشاركنورث كارولاينا: 6.06 ملغ / مل </td></tr><tr><td> آسيبرومازين </td><td> Vedco </td><td> VEDC207 </td><td> نهائي اضرب: 0،061 ملغ / مل </td></tr><tr><td> زيلازين </td><td> لويد </td><td> 4811 </td><td> نهائي اضرب: 0،667 ملغ / مل </td></tr><tr><td> Puralube الطبيب البيطري مرهم </td><td> Pharmaderm صحة الحيوان </td><td> 1621 </td><td></td></tr><tr><td> 0.9٪ حقن كلوريد الصوديوم جامعة جنوب المحيط الهادئ </td><td> باكستر </td><td> 2B1302 </td><td></td></tr><tr><td> 0.5 مم Microcurette </td><td> Roboz </td><td> RS-6350 </td><td></td></tr><tr><td> الكي الحراري وحدة </td><td> جيجر </td><td> 150-S/150A-S </td><td></td></tr><tr><td> TCU تلميح استبدال، حلقة الجميلة على التوالى </td><td> جيجر </td><td> 214 </td><td></td></tr><tr><td> مقص جراحي </td><td></td><td></td><td></td></tr><tr><td> التخزين المؤقت 10٪ محايد الفورمالين </td><td> سيغما </td><td> HT501128 </td><td></td></tr><tr><td> حمض الفورميك </td><td> الصياد </td><td> A119P-4 </td><td> خلط 426 مل من حمض الفورميك إلى 1047 مل ماء الصنبور، ثم يضاف 45 مل Rexyn 101 (H) </td></tr><tr><td> Rexyn 101 (H) </td><td> الصياد </td><td> R231-500 </td><td></td></tr><tr><td> الأنسجة تيك VIP E150/E300 </td><td> ساكورا </td><td></td><td></td></tr><tr><td> مشراح دوار </td><td> لايكا </td><td> RM2255 </td><td></td></tr><tr><td> ماير الهيماتوكسيلين </td><td> سيغما </td><td> MHS16-500ML </td><td></td></tr><tr><td> جيل NO.1 الهيماتوكسيلين </td><td> سيغما </td><td> GHS116 </td><td></td></tr><tr><td> يوزين B </td><td> سيغما </td><td> 2853 </td><td></td></tr><tr><td> Microtainer المصل فاصل </td><td> دينار بحريني Medical </td><td> 365956 </td><td></td></tr><tr><td> التخزين المؤقت فوسفات المالحة </td><td></td><td></td><td> 100 ملي مول ناه 2 PO 4، 140 مم كلوريد الصوديوم، ودرجة الحموضة 7.4 </td></tr><tr><td> البروتيني كوكتيل المانع، EDTA خالية </td><td> الحرارية العلمية </td><td> 78415 </td><td></td></tr></tbody></table>

examining the role of nasopharyngeal-associated lymphoreticular tissue (nalt) in mouse responses to vaccines

أنسجة البلعوم المرتبط اللمفي (NALT) وجدت في البشر، والقوارض، وغيرها من الثدييات، والمساهمة في الحصانة في الجيوب الأنفية 1-3. وNALT هما مواز على شكل جرس الهياكل الموجودة في الممرات الأنفية فوق الحنك الصلب، وعادة ما تعتبر أن تكون المكونات الثانوية للنظام المخاطية المرتبطة اللمفاوية و 4-6. تقع داخل NALT هي مقصورات منفصلة من الخلايا الليمفاوية B و T تتخللها مستضد تقديم الخلايا الجذعية 4،7،8. وتحاط هذه الخلايا من قبل طبقة الخلايا الظهارية مقحم مع M-الخلايا التي هي المسؤولة عن استرجاع مستضد من الأسطح المخاطية في الممرات الجوية 9،10. تستعد الخلايا الليمفاوية ساذج المتداولة خلال NALT للرد على اللقاءات الأولى مع مسببات الأمراض التنفسية 7. في حين NALT تختفي في البشر قبل سن سنتين، أجهزة فالداير اللمفاوي الطوق ومنظم على نحو مماثل لا تزال قائمة الroughout الحياة 6. وعلى النقيض من البشر والفئران الاحتفاظ NALT في جميع مراحل الحياة، وبالتالي توفير نموذج حيواني مناسب لدراسة الاستجابات المناعية تنشأ داخل الجيوب الأنفية 11. ثقافات وحيدة الخلية تعليق من NALT ليست عملية بسبب العائدات المنخفضة من خلايا وحيدات النوى. ومع ذلك، يمكن النظر NALT البيولوجيا بواسطة استزراع الأحياء السابقين من الجهاز سليمة، وهذا الأسلوب له ميزة إضافية للحفاظ على بنية الأنسجة الطبيعية. لفي الدراسات المجراة، ونماذج خروج المغلوب الوراثية تقديم العيوب تقتصر على NALT غير متوفرة حاليا بسبب وجود سوء فهم من المسار التنموي. على سبيل المثال، في حين سم لمفي-α الفئران خروج المغلوب وقد ضمر NALT، تتغير أيضا البقع باير، والعقد اللمفاوية الطرفية، والخلايا الجذعية والأنسجة اللمفاوية مسامي الأخرى في هذه الفئران التلاعب وراثيا 12،13. كبديل عن الفئران خروج المغلوب الجينات، والعمليات الجراحية ص الاجتثاثيلغي ermanently NALT من ممر الأنف دون أن يؤثر ذلك الأنسجة الأخرى. وقد استخدم نموذج الفأر مما أدى إلى إقامة علاقات بين استجابات NALT والمناعية للقاحات 1،3. جمع المسلسل من مصل الدم، ويغسل، لعاب الانف والافرازات المهبلية هو ضروري لوضع الأساس للاستجابات المضيف إلى تلقيح، في حين يمكن التأكد من الاستجابات المناعية المنشأ مباشرة من NALT بواسطة زراعة الأنسجة. الإجراءات التالية الخطوط العريضة لعملية جراحية، وزراعة الأنسجة وجمع العينات اللازمة لفحص المحلية والنظامية الاستجابات المناعية الخلطية للتلقيح (IN) داخل الأنف.

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Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

وصفت وسائل لدراسة مساهمات للأنسجة البلعوم المرتبط اللمفي (NALT) إلى الاستجابات المناعية الأنف والنظامية من الفئران لقاحات داخل الأنف. علينا أن نبرهن لعملية جراحية لوضع نموذج الفأر NALT التي تعتمد على و<em&gt; خارج الحي</em&gt; ثقافات NALT المستخرجة.

دراسة دور النسيج اللمفي البلعوم المرتبط (NALT) في الردود الفأر إلى اللقاحات

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

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Immunology and Infection

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

25.7K Views.  U.S. Army Medical Research Institute of Infectious Diseases. وصفت وسائل لدراسة مساهمات للأنسجة البلعوم المرتبط اللمفي (NALT) إلى الاستجابات المناعية الأنف والنظامية من الفئران لقاحات داخل الأنف. علينا أن نبرهن لعملية جراحية لوضع نموذج الفأر NALT التي تعتمد على و خارج الحي ثقافات NALT المستخرجة.

Video: Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

قياس الحمولة الجرثومية والاستجابات المناعية في الفئران المصابة المستوحدة الليستيريا</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

إدارة الأنف المؤتلف لقاحات الأنفلونزا في همي ماوس نماذج لدراسة الأغشية المخاطية الحصانة

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

عزل وتحليل العدلة لتحديد ما دورها في الغدد اللمفاوية حساسية الخلية وكلاء العلاجية

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

أوفيركسريسيون وتنقية الإنسان<em&gt; رابطة الدول المستقلة</em&gt; -prenyltransferase في<em&gt; الإشريكية القولونية</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

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. ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

تحصين الزرد الكبار للفحص السريري للقاحات الحمض النووي القائم

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

تقييم استجابات المضيف الممرض ونجاعة اللقاح في الفئران

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

منصة زرع نخاع العظم للتحقيق في دور الخلايا التطبيب في مرض الكسب غير المشروع مقابل المضيف

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

النقل عاليه الكفاءة من الضامة الابتدائية مع في المختبر كتب mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...