Njoku 박사는이 모델의 공식화를 초래 한 그의 지침과 통찰력있는 토론에 대해 Dr. Noel R. Rose, MD PhD를 인정하고 싶습니다.

<ol>
	<li>Castiella, A., Zapata, E., Lucena, M. I., Andrade, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+autoimmune+liver+disease:+A+diagnostic+dilemma+of+an+increasingly+reported+disease.">Drug-induced autoimmune liver disease: A diagnostic dilemma of an increasingly reported disease.</a> World J. Hepatology. 6 (4), 160-168 (2014).</li><li>Bjornsson, E. S., Bergmann, O. M., Bjornsson, H. K., Kvaran, R. B., Olafsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence,+presentation,+and+outcomes+in+patients+with+drug-induced+liver+injury+in+the+general+population+of+Iceland.">Incidence, presentation, and outcomes in patients with drug-induced liver injury in the general population of Iceland.</a> Gastroenterology. 144 (7), 1419-1425 (2013).</li><li>Castiella, A., Lucena, M. I., Zapata, E. M., Otazua, P., Andrade, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+autoimmune-like+hepatitis:+a+diagnostic+challenge.">Drug-induced autoimmune-like hepatitis: a diagnostic challenge.</a> Digestive Diseases and Sciences. 56 (8), 2501-2502 (2011).</li><li>Czaja, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+autoimmune-like+hepatitis.">Drug-induced autoimmune-like hepatitis.</a> Digestive Diseases and Sciences. 56 (4), 958-976 (2011).</li><li>Pohl, L. R., Thomassen, D., Pumford, N. R., Butler, L. E., Satoh, H., Ferrans, V. J., Perrone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hapten+carrier+conjugates+associated+with+halothane+hepatitis.">Hapten carrier conjugates associated with halothane hepatitis.</a> Advances in Experimental Medicine and Biology. 283, 111-120 (1991).</li><li>Njoku, D. B., Talor, M. V., Fairweather, D., Frisancho-Kiss, S., Odumade, O. A., 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+novel+model+of+drug+hapten-induced+hepatitis+with+increased+mast+cells+in+the+BALB/c+mouse.">A novel model of drug hapten-induced hepatitis with increased mast cells in the BALB/c mouse.</a> Experimental and Molecular Pathology. 78 (2), 87-100 (2005).</li><li>Cottagiri, M., Nyandjo, M., Stephens, M., Mantilla, J., Saito, H., Mackay, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+drug-induced,+immune-mediated+hepatitis,+interleukin-33+reduces+hepatitis+and+improves+survival+independently+and+as+a+consequence+of+FoxP3++T-cell+activity.">In drug-induced, immune-mediated hepatitis, interleukin-33 reduces hepatitis and improves survival independently and as a consequence of FoxP3+ T-cell activity.</a> Cellular and Molecular Immunology. , (2018).</li><li>Lohse, A. W., Manns, M., Dienes, H. P., Meyer zum Buschenfelde, K. H., Cohen, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+hepatitis:+disease+induction,+time+course+and+T-cell+reactivity.">Experimental autoimmune hepatitis: disease induction, time course and T-cell reactivity.</a> Hepatology. 11 (1), 24-30 (1991).</li><li>McCarthy, E. K., Vakos, A., Cottagiri, M., Mantilla, J. J., Santhanam, L., Thomas, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Shared+Cytochrome+p4502E1+Epitope+Found+in+Anesthetic+Drug-Induced+and+Viral+Hepatitis.">Identification of a Shared Cytochrome p4502E1 Epitope Found in Anesthetic Drug-Induced and Viral Hepatitis.</a> mSphere. 3 (5), (2018).</li><li>Cho, J., Kim, L., Li, Z., Rose, N. R., Talor, M. V., Njoku, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+bias+in+experimental+immune-mediated,+drug-induced+liver+injury+in+BALB/c+mice:+suggested+roles+for+Tregs,+estrogen,+and+IL-6.">Sex bias in experimental immune-mediated, drug-induced liver injury in BALB/c mice: suggested roles for Tregs, estrogen, and IL-6.</a> PLoS. One. 8 (4), 61186 (2013).</li><li>Satoh, H., Gillette, J. R., Takemura, T., Ferrans, V. J., Jelenich, S. E., Kenna, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+immunological+basis+of+halothane-induced+hepatotoxicity.">Investigation of the immunological basis of halothane-induced hepatotoxicity.</a> Advances in Experimental Medicine and Biology. 197, 657-673 (1986).</li><li>Eliasson, E., Kenna, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450+2E1+is+a+cell+surface+autoantigen+in+halothane+hepatitis.">Cytochrome P450 2E1 is a cell surface autoantigen in halothane hepatitis.</a> Molecular Pharmacology. 50 (3), 573-582 (1996).</li><li>Bourdi, M., Chen, W., Peter, R. M., Martin, J. L., Buters, J. T., Nelson, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytochrome+P450+2E1+is+a+major+autoantigen+associated+with+halothane+hepatitis.">Human cytochrome P450 2E1 is a major autoantigen associated with halothane hepatitis.</a> Chemical Research in Toxicology. 9 (7), 1159-1166 (1996).</li><li>Njoku, D. B., Li, Z., Washington, N. D., Mellerson, J. L., Talor, M. V., Sharma, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppressive+and+pro-inflammatory+roles+for+IL-4+in+the+pathogenesis+of+experimental+drug-induced+liver+injury.">Suppressive and pro-inflammatory roles for IL-4 in the pathogenesis of experimental drug-induced liver injury.</a> European Journal of Immunology. 39 (6), 1652-1663 (2009).</li><li>Aithal, G. P., Ramsay, L., Daly, A. K., Sonchit, N., Leathart, J. B., Alexander, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+adducts,+circulating+antibodies,+and+cytokine+polymorphisms+in+patients+with+diclofenac+hepatotoxicity.">Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity.</a> Hepatology. 39 (5), 1430-1440 (2004).</li><li>Higuchi, S., Kobayashi, M., Yoshikawa, Y., Tsuneyama, K., Fukami, T., Nakajima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-4+mediates+dicloxacillin-induced+liver+injury+in+mice.">IL-4 mediates dicloxacillin-induced liver injury in mice.</a> Toxicology Letters. 200 (3), 139-145 (2011).</li><li>Rubtsova, K., Marrack, P., Rubtsov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+autoimmunity.">Sexual dimorphism in autoimmunity.</a> Journal of Clinical Investigation. 125 (6), 2187-2193 (2015).</li><li>Satoh, H., Fukuda, Y., Anderson, D. K., Ferrans, V. J., Gillette, J. R., Pohl, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+studies+on+the+mechanism+of+halothane-induced+hepatotoxicity:+immunohistochemical+evidence+of+trifluoroacetylated+hepatocytes.">Immunological studies on the mechanism of halothane-induced hepatotoxicity: immunohistochemical evidence of trifluoroacetylated hepatocytes.</a> Journal of Pharmacology and Experimental Therapeutics. 233 (3), 857-862 (1985).</li><li>Habeeb, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+free+amino+groups+in+proteins+by+trinitrobenzenesulfonic+acid.">Determination of free amino groups in proteins by trinitrobenzenesulfonic acid.</a> Analytical Biochemistry. 14 (3), 328-336 (1966).</li><li>Christen, U., Burgin, M., Gut, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halothane+metabolism:+immunochemical+evidence+for+molecular+mimicry+of+trifluoroacetylated+liver+protein+adducts+by+constitutive+polypeptides.">Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.</a> Molecular Pharmacology. 40 (3), 390-400 (1991).</li><li>Christen, U., Quinn, J., Yeaman, S. J., Kenna, J. G., Clarke, J. B., Gandolfi, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+dihydrolipoamide+acetyltransferase+subunit+of+the+human+pyruvate+dehydrogenase+complex+as+an+autoantigen+in+halothane+hepatitis.+Molecular+mimicry+of+trifluoroacetyl-lysine+by+lipoic+acid.">Identification of the dihydrolipoamide acetyltransferase subunit of the human pyruvate dehydrogenase complex as an autoantigen in halothane hepatitis. Molecular mimicry of trifluoroacetyl-lysine by lipoic acid.</a> European Journal of Biochemistry. 223 (3), 1035-1047 (1994).</li></ol>

저자는 공개 할 것이 없습니다.

이 프로토콜의 강점은 재현성에 있습니다. 따라서 제안 된 단계를 준수하는 것이 중요합니다. 면역원의 제형은 일부에 대한 장벽이 될 수 있고; 그러나 우리는 문서에 설명 된 에피토프를 사용하여 모델을 재현하여 간장의 S100 분획을 분리 할 필요가 없습니다. 추가적인 에피토프 또는 단백질이 변경되어 면역화 후 간염을 유발할 수 있을 가능성이 높습니다. 그러나 우리는 신뢰할 수있는 결과와 ?...

이 방법의 목적은 생체 내에서 발생하는 약물 유도 자가면역 간염의 마우스 모델을 기술하고이 질병의 분자적, 면역 학적 및 유전 적 기초를 조사하는 데 어떻게 활용 될 수 있는지 입증하는 것입니다. 우리 연구의 장기적인 목적은 감수성 환자에서 DIH를 연구함으로써 만성 간 염증 및 부상의 발달을 담당하는 메커니즘을 밝히는 것입니다. 간 질환과 간경변은 25 세에서 64 세 사이의 성인에서 여섯 번째로 흔한 사망 원인을 구성합니다. 약물 유발 자가면역 간염(DIH)이라고도 하는 특발성 DILI는 미국에서 급성 간 기능 부전의 세 번째로 흔한 원인입니다. DIH는 자가면역간염 1형 간염 환자의 약 9~12%에서 관찰되는 가장 흔한 간 약물-유도성 과민증 과정이다. DIH 환자의 압도적 인 대다수는 여성 2,3,4입니다. DIH의 유형은 이소플루란, 세보플루란, 데스플루란 또는 할로탄과 같은 할로겐화 휘발성 마취제를 투여한 후 감수성이 있는 개체에서 발생합니다. 이러한 마취제는 신진 대사의 반응성 산물과 간 단백질에 공유적으로 결합하여 알레르기 또는 자가면역 반응을 유도 할 수있는 새로운 자기 항원을 만듭니다5.
마취제 및 DIH의 모든 형태의 개발에 관련된 병원성 메커니즘에 대한 연구는 이전에 인간 질병의 유도를 밀접하게 모방하는 동물 모델의 부족으로 인해 방해 받았다. 우리는 환자에서 면역 매개 DILI와 유사한 기능을 가진 DIH의 실험적 뮤린 모델을 개발했습니다. 간염은 시토크롬 P450 2E1(CYP2E1)5 효소에 의해 마취제의 산화 대사 후 형성되는 트리플루오로아세틸 클로라이드(TFA) 대사산물에 의해 공유적으로 변형된 두 개의 자가항원 중 하나로 면역화에 의해 유도된다. 하나의 자가항원은 여러 단백질6의 혼합물인 간 세포질 S100 간 분획이고, 두 번째 자가항원은 마취성 면역 매개 DILI7을 갖는 환자로부터 혈청에 의해 인식되는 CYP2E1의 에피토프이다. 실험적 자가면역간염에 비교적 내성이 있는 BALB/c 마우스를 사용하여 C57Bl/6J 마우스8의 자가면역간염의 S100 유도 면역화 모델과 모델을 구별합니다.
다양한 임상 프리젠 테이션 때문에 DIH는 환자에서 연구하기가 어렵습니다. 번역 실험 모델은 생체내 및 시험관 내에서 질병의 발병기전을 평가하는 능력을 제공한다. 현재, DIH를 유도하기 위한 다른 대안적인 방법은 동물의 사용 없이 생체내 또는 시험관내 적응성 또는 선천적 면역 반응을 완전히 검사하는 것이 아니다. 더욱이, S-100 또는 CYP2E1 에피토프의 트리플루오로아세틸화는 자극성 면역원을 생성하는 것으로 보이지 않으며, 우리는 TFA 변경된 단백질로 면역화함으로써 DIH를 유도하고 있기 때문에, 이들 동물은 면역화 또는 다른 절차 전에 에테르, 할로겐화 마취제, 바르비투레이트 또는 알코올을 투여받지 않을 것이며, 이러한 제제가 우리가 연구하고있는 파라미터를 바꿀 수 있다는 점을 고려한다. 그럼에도 불구하고, 우리는 컴퓨터 시뮬레이션을 활용하여 발견 된 CYP2E1 에피토프9 의 결합 선호도를 확인하고 여성 BALB / c 마우스가 더 심한 DIH10을 개발한다는 것을 보여줌으로써 여성의 성별을 암시하는 인간 DIH를 미러링함으로써 마우스 사용을 줄였습니다.
환자에서 DIH의 다양한 제시와 임상 질환 연구의 도전에도 불구하고, 반응성 약물 대사 산물에 의한 천연 단백질의 번역 후 변형은 할로겐화 마취제11을 따르는 DIH 병인에서 받아 들여지는 핵심 메커니즘입니다. 조사관들은 또한 CYP2E1이 이 과정12,13에서 주요 자가항원이라는 것을 받아들인다. 번역 후 변형된 CYP2E1 및 기타 간 단백질을 인식하는 인터루킨(IL)-4-상향조절된 CD4+T 세포의 역할은 호중구, 호산구 및 비만 세포를 간(14)으로 유인함으로써 마취 DIH의 수용된 개시제이며, 이 기전은 DIH15,16의 여러 형태에서 확인되었다. 유도된 FoxP3-발현 CD4+CD25+T 세포 (Tregs)는 DIH의 중증도를 감소시키고, 비장에서 이들 세포의 상대적 결핍은 DIH 10,7을 악화시킨다. 따라서, DIH를 이해하는 진보의 대부분은 생체내 및 시험관내 DIH의 유전적, 대사적 및 면역학적 메카니즘을 평가하기 위해 생체내 마우스 모델을 활용함으로써 가능하게 되었다.
우리와 다른 연구자들이 다른 마우스 모델을 사용하여 DIH를 개시하는 과정에서 IL-4, 호중구 및 호산구에 대한 역할을 밝혀 냈기 때문에이 관찰은 사용 된 DIH 모델에 관계없이 간염 및 부상이 IL-4에 의해 유도된다는 우리의 주장을 뒷받침한다고 믿습니다. 우리의 프로토콜의 강점은 수컷 및 암컷 마우스 모두의 생체 내 방법론의 활용과 조직학, CD4+ T 세포 증식 분석 및 사이토카인의 반복에 있습니다. 시험관 내 연구의 우리의 사용의 강점은 DIH를 유도하는 세포 상호 작용을 분리하는 방법론을 제공하면서 필요한 생쥐의 수를 줄인다는 것입니다. 우리는 수컷과 암컷 마우스의 사용을 권장하는데, 이는 결과의 해석에서 무의식적 편향의 가능성을 줄이고 DIH의 발생률, 유병률 및 중증도가 여성17에서 더 높기 때문에 우리 연구의 번역 잠재력을 강화하기 때문입니다. 마우스는 단일 공급 업체에서 얻는 것이 좋습니다. 그러나 이것이 가능하지 않다면 유전자 변형 마우스와 동일한 공급 업체로부터 깔짚 메이트 대조군 또는 야생형 마우스를 얻으십시오.

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			<td height="42" style="height:42px;width:216px;">0.1% 2,4,6-trinitrobenzene sulfonic acid (TNBS)</td>
			<td style="width:71px;">ThermoFisher</td>
			<td align="right" style="width:119px;">28997</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AKP Substrate Kit</td>
			<td style="width:71px;">BioRad</td>
			<td style="width:119px;">172-1063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BALB/c mice</td>
			<td style="width:71px;">Jackson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellTrace&#8482; CFSE Cell Proliferation Kit</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">C34554</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">CFA H37Ra</td>
			<td style="width:71px;">Becton Dickinson (Difco Bacto)</td>
			<td align="right" style="width:119px;">231131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FcR Blocking reagent</td>
			<td style="width:71px;">Milteyi</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">General supplement</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">HPRG770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HepaRG&#8482; cells cryopreserved</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">HPR GC10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Live/Dead Fixable Aqua Dead Cell stain kit&#160;</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">L34965</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaHC03</td>
			<td style="width:71px;">Millipore Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Percoll&#174;</td>
			<td style="width:71px;">Millipore Sigma</td>
			<td style="width:119px;">P1644-1L</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pertussis Toxin</td>
			<td style="width:71px;">List Biologicals</td>
			<td align="right" style="width:119px;">180</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate Buffered Saline pH 7.4</td>
			<td style="width:71px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce&#8482; Protease Inhibitor Mini Tablets, EDTA Free</td>
			<td style="width:71px;">ThermoFisher</td>
			<td align="right" style="width:119px;">88666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Hydroxide</td>
			<td style="width:71px;">JT Baker</td>
			<td style="width:119px;">3140-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">S-ethyltrifluorothioacetate (S-ETFA)</td>
			<td style="width:71px;">Millipore Sigma</td>
			<td align="right" style="width:119px;">177474</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Slide-a-lyzer dialysis cassettes (10 K, 12 ml)</td>
			<td style="width:71px;">ThermoFisher</td>
			<td align="right" style="width:119px;">66810</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UltraPure&#8482; SDS Solution, 10%</td>
			<td style="width:71px;">ThermoFisher</td>
			<td align="right" style="width:119px;">24730020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Williams Media E, no phenol red</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">A1217601</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of drug-induced, autoimmune hepatitis in balb/c mice for the study of its pathogenic mechanisms

약물-유도성 자가면역간염(DIH)은 자가면역간염 환자의 약 9~12%에서 관찰되는 가장 흔한 간약물 유도성 과민화 과정이다. DIH 환자의 압도적 인 대다수는 여성입니다. 유병률에서 이러한 성별 차이의 근본적인 메커니즘은 인간의 질병을 모방 한 동물 모델의 소박함 때문에 명확하지 않습니다. 그럼에도 불구하고, 근본적인 메카니즘은 인간 백혈구 항원 일배체형 및 성 호르몬과 관련되는 것으로 널리 믿어지고 있다. 대조적으로, DIH 마우스 모델을 사용하여, 우리는 IL-4 개시 CD4+ T 세포가 시토크롬 P450 2E1의 에피토프에 대해 지시된 것을 밝혀냈고, 이는 여성 BALB/c 마우스의 간으로의 호중구, 대식세포 및 비만 세포의 유입을 유도한다. 이 모델을 사용하여, 우리는 또한 IL-33-유도된 FoxP3+조절 T 세포가 암컷 및 수컷 마우스에서 DIH에 대한 보호를 부여한다는 것을 보여주었다. 이 DIH 모델은 DIH와 관련된 약물 대사 산물과 공유적으로 변경된 CYP2E1의 에피토프로 마우스를 면역화함으로써 유도된다. 이러한 에피토프는 DIH 환자에 의해 인식된다. 우리의 방법은 DIH의 발병 기전을 연구하는 데 활용할 수있는 강력하고 재현 가능한 간염 및 자가항체를 유도합니다. 생체내 연구가 부적절하게 행해질 때 마우스에서 과도한 통증과 고통을 야기할 수 있지만, 생체내 모델의 장점은 다수의 마우스에서 질병의 발병기전을 평가할 수 있다는 것이다. 또한, 변경된 간 단백질의 생물학적 효과는 침습적 인 절차를 사용하여 연구 할 수 있습니다. 시험관 내 연구를 실험 설계에 추가하면 세포 수준에서 신속한 반복 및 기계론적 분석이 가능합니다. 따라서, 우리는 우리의 모델 프로토콜과 DIH의 생체 내 및 시험관 내 메커니즘을 연구하는 데 어떻게 활용 될 수 있는지 입증 할 것입니다.

도 1에 도시된 DIH를 유도하기 위해 활용된 면역화 스케쥴은 목의 기저부(제0일제)와 꼬리의 기저부(제7일제)에서 요구되는 두 개의 면역화를 나타낸다. 도 2는 마취제의 CYP2E1, JHDN5, CYP2E1 에피토프 및 트리플루오로아세틸(TFA) 대사산물에 반응하여 CFSE를 사용하여 14일째에 수득된 대표적인 증식 데이터를 보여준다. 도 3은 14일째에 ?...

Watch this Scientific Journal Video about Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms at JoVE.com

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

우리는이 질병에서 볼 수있는 성별 차이를 포함하여 약물 유발 자가면역 간염의 발병 기전을 연구하는 데 활용할 수있는 BALB / c 마우스의 생체 내 예방 접종, 번역 형 간염 모델을 설명합니다. 우리는이 모델이 생체 내 및 시험관 내 실험 기술을 사용하여 재현 가능한 분석을 시연하는 방법을 설명 할 것입니다.

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 ...

induction-drug-induced-autoimmune-hepatitis-balbc-mice-for-study-its

Research

JoVE Journal

Immunology and Infection

2.9K 조회수.  Johns Hopkins University. 우리는이 질병에서 볼 수있는 성별 차이를 포함하여 약물 유발 자가면역 간염의 발병 기전을 연구하는 데 활용할 수있는 BALB / c 마우스의 생체 내 예방 접종, 번역 형 간염 모델을 설명합니다. 우리는이 모델이 생체 내 및 시험관 내 실험 기술을 사용하여 재현 가능한 분석을 시연하는 방법을 설명 할 것입니다.

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

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

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

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 ...

연구

면역학 및 감염병학

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 ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

인간의 장내 상피 세포와 BALB / C 마우스 대 식세포를 사용하여 웬만한 지방산과 콜레라 독소 농도의 결정

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

수초 희소 돌기 아교 세포 당 단백질 (MOG<sub&gt; 35-55</sub&gt;) C57BL / 6 쥐에 실험자가 면역 뇌척수염 (EAE) 유도

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

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

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

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

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

이펙터 CD4의 입양 전송하여 쥐과 장내 염증의 유도<sup&gt; +</sup&gt; CD45RB<sup&gt; 높은</sup&gt; 면역 결핍 마우스에게 T 세포

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

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

C 형 간염 바이러스에 대한 인간 락토페린의 세포 흡수 및 관련 바이러스 활동을 모니터링하는 면역 형광

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

B 형 간염 바이러스 리포터 시스템 개발은 복제주기의 초기 단계를 모니터링하기

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

복구 시간 증가 및 제한 염증 반응에서 미주 신경 자극 메커니즘을 연구에 LPS 관리를 감소

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

마비 및 비주얼 시스템 부상 Neuromyelitis Optica Autoantigen Aquaporin-4에 대 한 T 세포 관련 하 여 쥐에서의 유도

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...