이 작품은 PC에 NIH 부여 DK080351에 의해 지원되었다.

실험 프로토콜 이 실험 프로토콜의 첫 번째 단계는 마우스 뇌하수체의 땀샘의 다수를 수집하는 것입니다. 두 번째 단계는 단백질 추출물을 준비하기 위해 분비를 균질하는 것입니다. 세 번째 단계는 기름 혼합 이러한 단백질을 유상으로 만들다하는 것입니다. 1 단계 : 마우스 뇌하수체 분비의 수집과 저장 마우스 뇌하수체는 시신경 chiasm하여 삼차 신경 및 anteriorly로 옆으로 둘러싸인 쐐기 꼴의 뼈 셀라라는 turcica의 우울증에서 두개골 바닥에 앉아있다. 선은 큰 앞쪽에 엽과 작은 후부와 중간 엽 (叶)으로 구성하고, 평균 1.9 MG에 무게입니다. 뇌하수체 분비는 동물 보호 시설에 의해 euthanized 될 운명 혼합 변종, 성별 및 연령의 마우스에서 수집됩니다. 뇌하수체를 분리하려면, 마우스, 해부 두개골 위의 피부를 euthanized, 그리고 두개골의 뼈를 가위로 잘라 열려 있습니다. 두뇌는 다음 주변 두라 메이터으로부터 격리하는 뇌하수체를 노출 채취한 쐐기 꼴의 뼈 밖으로 훔쳐와 드라이 아이스에 플라스틱 튜브에 저장됩니다. 일반적으로 우리는 하나의 단백질 준비, 9 시간 만에 한 사람에 의해 수집된 수있는 번호는 300 마우스 뇌하수체 분비를 사용합니다. 일단 수집이 완료되면, 뇌하수체 분비가 들어있는 튜브 ° C 준비 때까지 다음 단계 -80에 저장됩니다. 2 단계. 마우스 뇌하수체 단백질의 추출 <ol><li> 15 ML 팰컨 튜브 (카탈로그 없음 352,059)을하는 균질화 버퍼를 추가 수집 매 100 뇌하수체 분비에 대한 버퍼 250 μL를 사용하여 얼음 욕조에 보관. 셀 (0.2 M)와 physiologic PH (7.4)의 세포질 내부 생리학 존재하는 것과 유사한 이온 강도의 버퍼를 사용합니다. 우리는 칼륨이 테아제 억제제 칵테일 (시그마 - 알드리치, 카탈로그 노 P8340 - 1ML)을 보충 (0.161 M, pH를 7.4) 호수 버퍼 사용합니다. </li><li> 바로 균질 전에 버퍼에 뇌하수체 분비를 추가하고, 다음 팔콘 튜브 하단의 Polytron 균 질기의 발전기 (5 mm 직경)를 놓으십시오. 얼음 목욕에서 유지하면서 부드럽게 위아래로 팰컨 튜브를 이동, 30 초 동안 최대 rpm으로 균질. 그런 다음, 1 분 얼음 homogenate를 놓습니다. 균질를 반복하고 냉각 두 번 더 단계를 반복합니다. 이 부드러운 균질의 세포를 휴식하고 핵을 방해하지 않고 cytosolic 단백질을 출시한다. </li><li> 핵, 손상되지 않은 조직 및 불용성 물질 (예 : 결합 조직 및 변성 단백질 단지 등) 제거하려면 4 10 분 ° C. 낮은 속도 (1,000g)에서 homogenate를 원심 분리기 </li><li> , 새로운 팰컨 튜브에 (지금 PNS, 포스트 - 원자력 뜨는라고도 함)를 전송 뜨는 펠렛은 그대로두고 돌보는하고, 얼음에 PNS를 저장합니다. </li><li> 균질화 버퍼의 원래 볼륨 (수집된 모든 100 뇌하수체 분비에 대한 버퍼 250 μL)에서 펠렛을 Resuspend, 상기 균질 위와 같이 원심 분리기, 다음 첫 번째와 두 번째 PNS를 결합. </li><li> -80에서 microcentrifuge 튜브 및 저장소로 나누어지는가 두 풀링 PNS 샘플 ° C 다음 단계에 대한 준비까지. 이 단백질 준비는 -80에서 저장할 수 있습니다 ° C 있지만 점차 그 힘을 looses, 그래서 우리가 준비로부터 6 개월 이내에 그것을 사용하는 것이 좋습니다. 겔 전기 영동으로 단백질 수율 및 농도 BCA 분석에 의해 (NO 피어스 카탈로그. 23,225), 그리고 단백질의 품질을 결정하는 작은 나누어지는을 사용합니다. 일반적으로, 우리는 40 MG / ML 주변 농도에서 균질화 버퍼의 1.5 ML 300 마우스 pituitaries에서 단백질의 약 60 MG를 구하십시오. </li></ol> 3 단계. 예방 접종에 대한 감광 유제의 준비 <ol><li> 빠르게 실험에 필요한 냉동 단백질 aliquots을 녹여 20 MG / ML에 단백질 농도를 조정합니다. 이 단백질 추출물 5 MG / Mycobacterium 결핵 (백톤 디킨슨, 231,141)를 죽이고 열을 ML을 포함하는 완전한 프로 인드의 도움이되는 (CFA, 시그마 F - 5881)로 1시 1분을 유화 것입니다. 각 마우스 에멀전 100 μL에 뇌하수체 추출의 1 밀리그램을 받게됩니다. </li><li> 여기에 주어진 예제는 각 마우스가 뇌하수체 단백질의 1 MG를 포함하는 유제 100 μL로 주입됩니다 10 마우스를 immunizing입니다. 준비하는 동안 재료의 손실의 두 가지 별도의 마우스를 허용하므로 뇌하수체 단백질 12 MG를 사용합니다. 20 MG / ML 뇌하수체 단백질 추출물의 바램이 600 μL은 2.5 ML 해밀턴 gastight의 주사기를 사용하여 위에서 설명합니다. 흔들림으로 CFA를 Resuspend, 다른 2.5 ML 해밀턴 주사기로 CFA 600 μL를 열망. 첨부와 CFA 가득한 주사기에 22 게이지 마이크로 emulsifying 바늘을 확보. 천천히 바늘이 CFA으로 가득합니다 이러한 CFA의 주사기의 플런저를 밀어 후 첨부의 다른 쪽 끝을 확보뇌하수체 추출 가득 주사기에 바늘. </li><li> 천천히 단지 소량이 혼합되어 같은 acqueous 뇌하수체 추출물로 오일 구성 요소를 밀어 CFA의 주사기의 플런저를 전진. 다음 CFA의 주사기에 뇌하수체 추출 주사기의 플런저를 뒤로 밀어. 점차적으로 혼합 물질의 양을 증가이 작업을 반복합니다. 두 주사기의 전체 내용이 혼합 때까지 계속합니다. 당신은 물 - 인 - 오일 에멀젼을 나타내는 약간 흰하고 고르게 분산 솔루션을 얻을 것입니다. </li><li> 유제가 형성되면, 유제가 완전히 혼합되도록 적어도 500 번 백업 및 앞뒤 움직임을 반복합니다. 최종 혼합 사이클 후 1시 1분 물 - 인 - 오일 에멀젼 사용 준비가되어 10 MG / ML의 뇌하수체 단백질 농도가 포함되어 있습니다. 유제가로 동반자 조브 문서에서 자세한 내용, subcutaneously anesthetized SJL 마우스로 당일에 주입됩니다. </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>

관심 없음 충돌 선언하지 않습니다.

면역 hypophysitis와 함께 첫 번째 환자는 1962 4보고했다는 사실에도 불구하고, 병원성 뇌하수체 autoantigens 5를 식별하기 위해 남아있다. 동물 모델은 환자 치료에 번역 아르 생체의 발견을 은닉이 autoantigens을 파악하는 것은 매우 유용할 수 있습니다. 이 문서는 실험 동물에서 면역 hypophysitis를 유도에 적합 형태로 뇌하수체 단백질을 준비 간단한 절차를 시연했다.

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<td>15-ml Falcon tubes</td>
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<td>homogenization buffer</td>
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<td>phosphate buffered saline</td>
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<td>protease inhibitor cocktail</td>
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<td>Polytron homogenizer</td>
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preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis

면역 hypophysitis가 발생하거나 autoimmunity 1과 함께 뇌하수체의 만성 염증이다. 그것은 전통적으로 희귀한 질병으로 간주 되었으나보고 최근 몇 년 동안 현저하게 증가하고 있습니다. Hypophysitis는 사실, 이러한 CTLA - 4 2, PD - 1 수용체와 같은 T lymphocytes에 표현 억제 수용체를 차단하는 항체로 치료 암 환자의 &#39;부작용&#39;로 쓰시하지 개발하고 있습니다. 면역 hypophysitis는 도움이되는 3 혼합 뇌하수체 단백질과 쥐를 주사하여 실험적으로 유도된 수 있습니다. 이 동영상이 문서에서 우리는 마우스 뇌하수체 분비 및 SJL 생쥐의 면역 hypophysitis를 유도에 적합한 형태로 그들을 준비하는에서 단백질을 추출하는 방법을 보여줍니다.

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

면역 hypophysitis는 마우스 뇌하수체의 단백질 추출물을 주사로 쥐 복제하​​실 수 있습니다.

실험 면역 Hypophysitis의 유도를위한 마우스 뇌하수체 Immunogen의 준비

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 조회수.  The Johns Hopkins University. 면역 hypophysitis는 마우스 뇌하수체의 단백질 추출물을 주사로 쥐 복제하​​실 수 있습니다.

비디오: 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 마우스의 실험적 면역 Hypophysitis의 유도

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

연구

면역학 및 감염병학

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.

효소 다이제스트 및 그라데이션 분리를 사용하여 마우스 피부에서 침투 백혈구의 분리

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.

다양한 조직에서 면역 세포의 질병에 의존하는 유통의 마우스 및 평가에 실험자가 면역 뇌척수염의 유도

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.

실험자가 면역 뇌척수염 연구 간단하고 효율적인 생산 및 마우스 미엘린 희소 돌기 아교 세포 당 단백질의 정제

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.

병원성 메커니즘 연구를위한 BALB / c 마우스에서 약물 유도,자가 면역 간염의 유도

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.

실험적 자가면역 포도막염: 안구내 염증성 마우스 모델

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.

실험적 자가면역 뇌척수염 유도를 위한 항원/보조 에멀젼을 제조하기 위한 빠르고 간단하며 표준화된 균질화 방법

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.

마우스 자발성 자가면역 갑상선염 모델 생성

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

자가면역뇌척수염에서 Mycobacterium paratuberculosis에 의한 면역원성 향상

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.

저자 스포트라이트: 실험적 자가면역 뇌척수염 중 자가항원의 면역원성 향상에 대한 Mycobacterium paratuberculosis의 보조 활성  

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.

실험적 자가면역 뇌척수염 동안 자가항원의 면역원성을 향상시키는 Mycobacterium paratuberculosis 의 보조 활성

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