这项工作是由美国国立卫生研究院授予DK080351 PC支持。

实验协议在这个试验性的协议的第一步是收集了大量鼠标脑垂体。第二个步骤是，同质化的腺体准备蛋白提取物。第三步是油性混合物乳化这些蛋白质。 第1步：鼠标脑垂体收集和储存鼠标脑下垂体位于颅底三叉神经和视交叉前方横向包围，在所谓蝶鞍蝶骨的抑郁症。腺体组成一个较大的前叶和较小的后部和中间叶，体重平均为1.9毫克。注定要被实施安乐死的动物护理设施的混合菌株，性别，和年龄，老鼠的脑垂体收集。 要隔离脑垂体，鼠标是安乐死，上述解剖颅骨的皮肤，并用剪刀剪开颅骨骨开放。解除大脑，脑垂体，然后从周围硬脑膜分离暴露，捞起来蝶骨上干冰的塑料管中。 一般来说，我们使用一个蛋白质的准备，可以由一人在9个小时收集的300鼠标脑垂体。 一旦收集完成，包含脑垂体管保存于-80 ° C，直到准备下一个步骤。 第2步。小鼠垂体蛋白的提取<ol><li>同质化的缓冲区，以添加15毫升猎鹰管（目录没有352059）保持在冰浴，每收集100脑垂体使用250的缓冲液。使用的是细胞质内的细胞生理pH（7.4）（0.2 M）和生理上的一个类似的离子强度缓冲。我们用磷酸盐缓冲液（0.161 M，pH值7.4），辅以一种蛋白酶抑制剂的鸡尾酒（Sigma - Aldrich公司，目录没有P8340 - 1ML）。 </li><li>添加脑垂体刚刚在同质化的缓冲区，然后将宝创匀浆发电机猎鹰管的底部（直径5毫米）。均质最高转速为30秒，轻轻移动猎鹰管向上和向下，同时保持在冰浴。然后休息1分钟冰匀浆。重复的同质化和冷却步骤两次。要打破这种温柔的同质化细胞，而不破坏的原子核并释放胞浆蛋白。 </li><li>要去除细胞核，完整的组织和不溶物（如结缔组织，变性的蛋白复合物），离心机在低转速（千克）的匀浆液在4 ° C 10分钟</li><li>转移上清（现在叫后核上清，PNS）到一个新的猎鹰管，照顾离开颗粒不受干扰，并存储在冰上的PNS的。 </li><li>在同质化的缓冲区的原始卷（250μL每收集100脑垂体的缓冲区）重悬沉淀，上述均质，离心上述，然后结合第二PNS的第一个。 </li><li>分装在两个汇集PNS的样品到离心管中并储存在-80 ° C直到为下一步做好准备。这种蛋白制备可存放于-80 ° C，但逐渐失去其效力，因此，我们建议在6个月从筹备内使用。使用一个小等分，以确定蛋白质产量和浓度由BCA检测（皮尔斯目录。23225），并通过凝胶电泳的蛋白质质量。在一般情况下，我们从300鼠标垂体获得约60毫克的蛋白质，同质化的缓冲液在1.5毫升，在浓度40毫克/毫升左右。 </li></ol>第3步。免疫乳液的制备<ol><li>迅速解冻实验所需的冷冻蛋白等分，调整蛋白浓度为20毫克/毫升。将这种蛋白质提取物乳化1:1与完全弗氏佐剂（终审法院，Sigma公司的F - 5881）含有5毫克/毫升杀害结核分枝杆菌（Becton Dickinson公司，231141）的热量。每个鼠标将获得1毫克的垂体提取物在100μL乳液。 </li><li>这里给出的例子是免疫10只小鼠，其中每个鼠标将注入100μL含有1毫克垂体蛋白质的乳液。允许两个额外的小鼠物质损失，所以在准备使用12毫克垂体蛋白质。上面描述的使用2.5毫升汉密尔顿气密性注射器的Aspire 600μL的20毫克/毫升垂体蛋白提取物。重悬通过晃动终审法院，并渴望进入另一个2.5 mL的汉密尔顿注射器的600μL的终审法院。连接和安全的一个22号的微乳化针头与终审法院填补了注射器。慢慢推针是与终审法院填补的终审法院注射器的柱塞，然后连接和安全的另一端充满垂体提取物的注射器针头。 </li><li>慢慢推进终审法院的注射器的柱塞推入acqueous脑垂体提取物，这样，只有少量混合油成分。然后向后推到终审法院的注射器柱塞的脑垂体中提取注射器。逐步增加混合材料的数量，重复此动作。继续混合，直到两个注射器的全部内容。您将获得代表的油包水型乳化发白和均匀分布的解决方案。 </li><li>一旦形成乳液，至少有500次重复的往复运动，以确保完全混合乳液。最终混合周期后，1:1的油包水型乳化液就可以使用，包含垂体蛋白浓度为10毫克/毫升。乳化将在同一天注射，皮下麻醉SJL小鼠，在同伴的Jove文章详细。 </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>

尽管自身免疫性垂体炎的第一个病人是在1962 年第4期报道，垂体自身抗原的致病性仍有待确定5。动物模型是非常有用的，以确定这些自身抗原的，协助的生物标志物，是可翻译的病人护理的发现。本文演示了一个简单的程序，准备在垂体蛋白质的形式，在实验动物诱发自身免疫性垂体炎是足够的。

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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

自身免疫性垂体炎是一种慢性炎症引起或伴随着自身免疫 1的脑下垂体。它历来被认为是一种罕见的疾病，但报告已在近年来显着增加。垂体炎，事实上，发展不寻常在块上，如CTLA - 4和PD - 1受体的T淋巴细胞，表达抑制性受体与抗体治疗癌症患者的一个“副作用“。实验小鼠注射佐剂3混合垂体蛋白质可诱发自身免疫性垂体炎。在这个视频文章中，我们演示了如何从鼠标脑垂体和如何准备他们在适合SJL小鼠诱导的自身免疫性垂体炎的形式提取的蛋白质。

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

可以复制自身免疫性垂体炎在小鼠体内，小鼠垂体蛋白提取物注射。

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

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 次观看.  The Johns Hopkins University. 可以复制自身免疫性垂体炎在小鼠体内，小鼠垂体蛋白提取物注射。

视频: 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.

SJL小鼠诱导实验性自身免疫性垂体炎

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

科研

免疫与感染

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.

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

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.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

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.

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

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.

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

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.

实验性自身免疫性葡萄膜炎：眼内炎症小鼠模型

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.

一种快速、简单、标准化的均质化制备抗原/辅助乳剂以诱导实验性自身免疫性脑脊髓炎的方法

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.

小鼠自发性自身免疫性甲状腺炎模型的产生

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

副结核分枝杆菌在自身免疫性脑脊髓炎中的免疫原性增强

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.

作者聚焦： 副结核分枝杆菌在增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原性中的辅助活性  

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.

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

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