وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح DK080351 إلى الكمبيوتر.

البروتوكول التجريبي الخطوة الأولى في هذا البروتوكول التجريبي هو جمع عدد كبير من الغدد النخامية الماوس. الخطوة الثانية هي التجانس الغدد إعداد استخراج البروتين. الخطوة الثالثة هي لاستحلب هذه البروتينات مع المزيج الزيتي. خطوة 1 : جمع وتخزين الغدد النخامية الماوس الغدة النخامية الماوس يجلس في قاعدة الجمجمة في منخفض من العظم الوتدي تسمى السرج التركي ، وتحيط بها أفقيا والأعصاب الثلاثي التوائم الأمامية قبل chiasm البصرية. وتتألف الغدة من الفص الأمامي أكبر وأصغر الفصوص الخلفية والمتوسطة ، ويزن 1.9 ملغ في المتوسط. وتجمع الغدد النخامية من الفئران من سلالات مختلطة من الجنسين ، والأعمار ، والمتجهة إلى يتم التخلص من الحيوانات مرفق الرعاية. لعزل الغدة النخامية ، والموت الرحيم للفأرة ، والجلد فوق تشريح الجمجمة وعظام الجمجمة قطع مفتوحة مع المقص. رفع الدماغ للكشف عن الغدة النخامية ، وهي معزولة ثم من الأم الجافية المحيطة بها ، وحصد من العظم الوتدي وتخزينها في أنبوب من البلاستيك على الثلج الجاف. بشكل عام نحن نستخدم الماوس الغدد النخامية 300 لإعداد بروتين واحد ، وهو الرقم الذي يمكن تحصيلها من قبل شخص واحد في 9 ساعات. بمجرد الانتهاء من جمع وتخزين أنبوب يحتوي على الغدد النخامية عند -80 درجة مئوية لتصبح جاهزة للخطوة التالية. الخطوة 2. استخراج البروتينات النخامية الماوس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أبقى إضافة إلى التجانس العازلة أنبوب فالكون 15 مل (أي 352059 التسويقي) في حمام الثلج ، وذلك باستخدام 250 ميكرولتر من العازلة لكل الغدد النخامية 100 التي تم جمعها. استخدام القوة العازلة الأيونية مماثلة لتلك التي موجودة من الناحية الفسيولوجية داخل سيتوبلازم الخلية (0.2 م) والرقم الهيدروجيني الفسيولوجي (7.4). نستخدم الفوسفات مخزنة المالحة (0،161 م ، ودرجة الحموضة 7.4) ، على أن تستكمل مع مثبط البروتياز الكوكتيل (سيغما الدريخ ، وأنواع لا P8340 - 1ML). </li><li style=";text-align:right;direction:rtl"> إضافة إلى الغدد النخامية العازلة قبل التجانس ، ثم ضع مولد (5 ملم قطر) من الخالط Polytron في الجزء السفلي من الأنبوب من طراز فالكون. التجانس في الدقيقة كحد أقصى لمدة 30 ثانية ، وتتحرك بلطف أنبوب فالكون صعودا وهبوطا مع إبقائها في حمام جليدي. ثم ، ضع جناسة على الجليد لمدة 1 دقيقة. كرر الخطوات من التجانس والتبريد مرتين أخريين. ينبغي لهذا التجانس طيف كسر الخلايا والافراج عن البروتينات عصاري خلوي دون تعطيل النوى. </li><li style=";text-align:right;direction:rtl"> لإزالة النوى ، والأنسجة والمواد غير القابلة للذوبان متواصلة (مثل النسيج الضام ومجمعات البروتين المعطل) ، أجهزة الطرد المركزي لجناسة بسرعة منخفضة (1000g) لمدة 10 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> نقل طاف (التي تسمى الآن في مرحلة ما بعد النووية الطافي (Supernatant) ، PNS) إلى أنبوب فالكون الجديدة ، مع الحرص على ترك بيليه دون عائق ، وتخزين PNS على الجليد. </li><li style=";text-align:right;direction:rtl"> Resuspend الكرية في حجم الأصلي للتجانس العازلة (250 ميكرولتر من العازلة لكل الغدد النخامية التي جمعت 100) ، على النحو الوارد أعلاه التجانس ، الطرد المركزي على النحو الوارد أعلاه ، ومن ثم الجمع بين PNS الثاني مع الأول. </li><li style=";text-align:right;direction:rtl"> قسامة اثنين تجميع العينات في أنابيب PNS microcentrifuge وتخزينها في -80 درجة مئوية لتصبح جاهزة للخطوة التالية. يمكن تخزين هذا البروتين في إعداد -80 درجة مئوية ولكن يفقد فعاليته تدريجيا ، ولذا فإننا نوصي لاستخدامه في غضون 6 أشهر من التحضير. استخدام قسامة صغيرة لتحديد العائد من البروتين والتركيز من قبل مقايسة BCA (بيرس كتالوج رقم 23225) ، ونوعية البروتين هلام إستشراد. بشكل عام ، ونحن الحصول على نحو 60 ملغ من البروتين من 300 pituitaries الماوس في 1.5 مل من التجانس العازلة ، بتركيز حوالي 40 ملغ / مل. </li></ol> الخطوة 3. إعداد مستحلب للتمنيع <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ذوبان الجليد بسرعة aliquots البروتينات اللازمة لتجميد التجربة وضبط تركيز البروتين إلى 20 ملغ / مل. وسيتم استخراج هذا البروتين مستحلب 01:01 فرويند الكامل مع المصاحب لل(CFA ، سيجما F - 5881) تحتوي على 5 ملغ / مل من الحرارة قتل المتفطرة السل (بيكتون ديكنسون ، 231141). وسوف تتلقى كل الماوس 1 ملغ من مستخلص الغدة النخامية في 100 ميليلتر من مستحلب. </li><li style=";text-align:right;direction:rtl"> على سبيل المثال هنا هو إعطاء لتحصين الفئران 10 ، حيث سيتم حقن كل الفأر مع 100 ميكرولتر من مستحلب تحتوي على 1 ملغ من البروتينات في الغدة النخامية. تسمح لاثنين الفئران اضافية عن الخسائر المادية أثناء إعداد ، وذلك باستخدام 12 ملغ من البروتينات في الغدة النخامية. يطمح 600 ميكرولتر من 20 ملغ / مل استخراج البروتين النخامية وصف أعلاه باستخدام 2.5 مل حقنة gastight هاملتون. Resuspend اتفاق وقف اطلاق النار من جانب والهز ، ويطمح 600 ميكرولتر من CFA إلى آخر حقنة هاميلتون 2.5 مل. إرفاق وتأمين عيار 22 الصغرى الاستحلاب الإبرة إلى حقنة مليئة CFA. دفع ببطء المكبس من المحاقن CFA بحيث يتم تعبئة الإبرة مع CFA ، ثم نعلق وتأمين الطرف الآخر منالإبرة إلى حقنة مليئة استخراج الغدة النخامية. </li><li style=";text-align:right;direction:rtl"> يتقدم ببطء المكبس من المحاقن CFA لدفع النفط الى عنصر استخراج الغدة النخامية acqueous ، بحيث يتم خلط كمية صغيرة فقط. ثم دفع المكبس الخلفي من الحقنة استخراج الغدة النخامية في حقنة CFA. كرر هذا الإجراء زيادة تدريجية في كمية المواد المختلطة. تستمر حتى يتم خلط المحتوى بالكامل من المحاقن اثنين. سوف تحصل على حل بيضاء وزعت بالتساوي الذي يمثل الماء في مستحلب النفط. </li><li style=";text-align:right;direction:rtl"> بمجرد أن يتم تشكيل مستحلب ، كرر الحركة ذهابا وإيابا على الأقل 500 مرة لضمان خلطها بشكل تام المستحلب. بعد دورة خلط النهائي ، 01:01 المياه في ومستحلب النفط جاهزة للاستخدام وتحتوي على تركيز البروتين النخامية من 10 ملغ / مل. وسيتم حقن مستحلب في اليوم نفسه تحت الجلد في الفئران SJL تخدير ، على النحو المفصل في المادة الرفيق إن الرب. </li></ol>

<ol>
	<li>Caturegli, P., Newschaffer, C., Olivi, A., Pomper, M. G., Burger, P. C., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Autoimmune Hypophysitis Endocr Rev. 26, 599-614 (2005).</li><li>Blansfield, J. A., Beck, K. E., Tran, K., Yang, J. C., Hughes, M. S., Kammula, U. S., Royal, R. E., Topalian, S. L., Haworth, L. R., Levy, C., Rosenberg, S. A., Sherry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T-lymphocyte-associated+antigen-4+blockage+can+induce+autoimmune+hypophysitis+in+patients+with+metastatic+melanoma+and+renal+cancer.">Cytotoxic T-lymphocyte-associated antigen-4 blockage can induce autoimmune hypophysitis in patients with metastatic melanoma and renal cancer.</a> J Immunother. 28, 593-598 (2005).</li><li>Tzou, S. C., Lupi, I., Landek, M., Gutenberg, A., Tzou, Y. M., Kimura, H., Pinna, G., Rose, N. R., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+Hypophysitis+of+SJL+mice:+Clinical+Insights+from+a+New+Animal+Model.">Autoimmune Hypophysitis of SJL mice: Clinical Insights from a New Animal Model.</a> Endocrinology. 149, 3461-3469 (2008).</li><li>Goudie, R. B., Pinkerton, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+hypophysitis+and+Hashimoto's+disease+in+a+woman.">Anterior hypophysitis and Hashimoto's disease in a woman.</a> J Pathol Bacteriol. 83, 584-585 (1962).</li><li>Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hypophysitis:+an+underestimated+disease+in+search+of+its+autoantigen(s).">Autoimmune hypophysitis: an underestimated disease in search of its autoantigen(s).</a> J Clin Endocrinol Metab. 92, 2038-2040 (2007).</li></ol>

الإعلان عن أي تضارب في المصالح.

على الرغم من حقيقة ان تم الابلاغ عن اول مريض مع التهاب النخامية الذاتية في عام 1962 4 ، والمسببة للأمراض الغدة النخامية autoantigens لا يزال يتعين تحديدها 5. نماذج حيوانية يمكن أن تكون مفيدة جدا لتحديد هذه autoantigens ، والمساعدة في اكتشاف المؤشرات الحيوية التي هي ترجم?...

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<td>15-ml Falcon tubes</td>
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preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis

التهاب النخامية الذاتية هو التهاب مزمن في الغدة النخامية أو بسبب المناعة الذاتية مصحوبة 1. وقد جرت العادة على اعتبار أنه مرض نادر ولكن التقارير قد ازداد بشكل ملحوظ في السنوات الأخيرة. التهاب النخامية ، في الواقع ، لم يتطور بدرجة غير عادية باعتبارها &quot;الآثار الجانبية&quot; في علاج مرضى السرطان مع الأجسام المضادة التي تعمل على منع المستقبلات المثبطة وأعرب عن الخلايا اللمفية تي ، مثل CTLA - 4 2 ومستقبلات PD - 1. يمكن أن يتسبب التهاب النخامية الذاتية تجريبيا عن طريق حقن الفئران مع البروتينات النخامية مختلطة مع مساعد 3. في هذه المقالة الفيديو نظهر كيفية استخراج البروتينات من الغدد النخامية الماوس وكيفية التحضير لها في شكل مناسب لإحداث التهاب النخامية في الفئران SJL الذاتية.

Watch this Scientific Journal Video about Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis at JoVE.com

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

يمكن استنساخها الذاتية التهاب النخامية في الفئران عن طريق حقن مستخرج من البروتينات النخامية الماوس.

إعداد مستمنع ماوس النخامية للتحريض التهاب النخامية الذاتية التجريبي

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

preparation-mouse-pituitary-immunogen-for-induction-experimental

Research

JoVE Journal

Immunology and Infection

14.4K Views.  The Johns Hopkins University. يمكن استنساخها الذاتية التهاب النخامية في الفئران عن طريق حقن مستخرج من البروتينات النخامية الماوس.

Video: Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

تحريض التهاب النخامية الذاتية التجريبية في الفئران SJL

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

عزل التسلل الكريات البيض من ماوس الجلد عن طريق الأنزيمية دايجست والفصل التدرج

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

تحريض التجريبية المناعة الذاتية التهاب الدماغ في الفئران وتقييم توزيع الأمراض التي تعتمد على خلايا المناعة في الأنسجة المختلفة

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

إنتاج بسيطة وفعالة وتنقية ماوس المايلين دبقية قليلة التغصن البروتين السكري لالتجريبية المناعة الذاتية الدراسات التهاب الدماغ

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

تحريض التهاب الكبد المناعي الذاتي الناجم عن الأدوية في الفئران BALB / c لدراسة آلياته المسببة للأمراض

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

التهاب القزحية المناعي الذاتي التجريبي: نموذج فأر التهابي داخل العين

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

طريقة تجانس سريعة وبسيطة وموحدة لإعداد مستحلبات المستضد / المستحلبات المساعدة لإحداث التهاب الدماغ والنخاع المناعي الذاتي التجريبي

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely ...

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

جيل من الفأر نموذج التهاب الغدة الدرقية المناعي الذاتي التلقائي

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

تعزيز الاستمناع بواسطة المتفطرة نظيرة السل في التهاب الدماغ والنخاع المناعي الذاتي

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

تسليط الضوء على المؤلف: النشاط المساعد للمتفطرة نظيرة السل في تعزيز مناعة المستضدات الذاتية أثناء التهاب الدماغ والنخاع المناعي الذاتي التجريبي  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

النشاط المساعد للمتفطرة نظيرة السل في تعزيز مناعة المستضدات الذاتية أثناء التهاب الدماغ والنخاع المناعي الذاتي التجريبي

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...