作者想感谢他们的技术援助阿曼达Tardo，博士穆斯塔法Bouljihad，斯蒂芬妮费力，卡罗尔柯尼博士艾琳Ribka，博士皮特迪迪埃和多萝西Kuebler。这项工作得到了NIH的拨款R01 - DK076653 DK076653 3R01 - 02S1金水TNPRC RR000164基地经营授予。

1。皮肤活检样品采集<ol><li>之前皮肤活检手术，麻醉动物肌注2.5毫克/公斤盐酸tiletamine zolazepam盐酸telazol的混合物（道奇堡动物健康，道奇堡，IA）。监控管理，直到斜卧麻醉的动物，然后删除其外壳。 </li><li>取下的头发和皮肤面积大小40刀片（奥斯特的专业产品，麦克明维尔，TN）利用奥斯特金A5单速兽医帆船利益无菌准备与优碘擦洗，酒精交替。一个在选定的活检部位的安全无菌孔的悬垂。 </li><li>使用无菌技术，一个4.0毫米Miltex打孔真皮活检仪器（Miltex，纽约，PA）对皮肤，同时旋转180度顺时针和逆时针轻微的压力的仪器，直到活检穿透真皮层断面进入皮下TIssue。取出活检样本和掌握钳和皮下组织的皮肤横断部分。 </li><li>一个十字图案附着在一个3 / 8圈切割针（Ethilon，爱惜康，强生公司医疗有限公司，伯克郡，英国）用3-0尼龙缝合关闭皮肤缺损。给所有的动物0.01毫克/千克手术后镇痛丁丙诺啡盐酸（Hospira公司，森林湖，IL）肌注。 </li></ol> 2。样品处理<ol><li>恒河猴慢性皮炎和健康控制工作，从皮肤活检标本。获得两到三个（4毫米直径）活检从每个动物的样本。 </li><li>锌福尔马林（Z轴修复，Anatech有限公司，巴特尔克里克，心肌梗死），24小时修复第一个范例，在水冲洗30分钟，简单地在70％乙醇洗，并将其放置到ASP300徕卡组织处理器（莱卡微系统公司水牛城树丛，KS）组织脱水70％，80％，95％和100％的乙醇48升等级每分钟（费希尔科学，宾夕法尼亚州匹兹堡），其次是两个对二甲苯的变化（费舍尔）。 </li><li>长期储存在室温下嵌入石蜡媒体（费舍尔）。广场前20分钟到切片在20℃。准备用旋转切片机（HM325，MICROM国际，华尔道夫，德国）6微米的部分。广场路段收取的幻灯片（费舍尔）和空气干燥，60℃过夜。染色与苏木精和伊红（H＆E）的标准方法（如下所述）。 </li><li>修正了2％多聚甲醛（USB公司，克利夫兰，俄亥俄州）在室温下30分钟的第二个样本，在磷酸盐洗三次缓冲液（PBS，GIBCO Invitrogen公司，卡尔斯巴德，加利福尼亚州），在30％的蔗糖（费舍尔） 4小时，并嵌入在华侨城冻结介质（樱花Finetek，托伦斯，CA）。前20分钟到切片- 80℃保持。使用低温恒温器（HM560，Thermo Scientific的卡拉马祖，心肌梗死），准备15微米的部分。 </li></ol> 3。 H＆E染色<ol><li> Deparaffinize嵌入DED部分：通过三个转变二甲苯（费舍尔）和补充水分，通过分级ethanols三个转变为100％，95％的两个变化，一个变化的80％和蒸馏水，每2分钟。 </li><li>染色苏木（理查德艾伦科学，卡拉马祖MI）1分钟，并遵循一个简单的洗，在运行自来水。 </li><li> （Sigma公司）与曙红染液为6分钟。 </li><li>通过95％和无水乙醇脱水染色组织，两个转变，每次2分钟，然后在三个变化对二甲苯明确，每3分钟。 </li><li>山玻璃罩使用Cytoseal安装介质（理查德 - 阿兰科学）在H＆E -染色组织切片。 </li></ol> 4。病理组织学评价检查H＆E -染色活检皮肤下的光显微镜使用4部分现货透视数码相机（数码仪器公司，美国密歇根州） - 40倍的放大倍率和徕卡显微镜。 5。免疫荧光STAI宁<ol><li> OCT包埋免疫荧光染色的皮肤部分的工作。染色前，浸泡20分钟PBS中含0.1％，0.2％的鱼皮明胶（FSG，Sigma公司）TRITON X - 100（Sigma公司）删除华侨城通透组织。 </li><li> 10％正常山羊血清（NGS，Sigma）的PBS - FSG稀释为1小时，在室温下，，孵育，洗涤后的幻灯片，在加湿的幻灯片室。 </li><li>为了形象化沉积的IgA和tTG2抗体，孵育反恒河猴IgA和tTG2初级抗体在10％农工商PBS - FSG稀释为1小时，幻灯片。 </li><li>洗10分钟，10分钟，PBS - FSG PBS - FSG - TRITON X - 100（Sigma公司）。 </li><li>孵育中学，同种特定标签的Alexa Fluor荧光488（绿色），568（红色）或633（蓝色）分子探针（Invitrogen公司）1:1,000稀释的抗体。使用TOPRO - 3染料可视化的核DNA。使用10％的农工商PBS - FSG作为抗体稀释液。 </li><li>使用反淬火液（Sigma公司），装入染色组织切片。 </li></ol> 6。共焦成像执行了Leica TCS SP2真正的共聚焦激光扫描显微镜配备了三个激光器（六行激发可用）（莱卡，韦茨拉尔，德国）成像：氩，氪激光波长为488（绿色），在568 nm的氪激光（红色）和在633 nm（蓝色），从可见光谱的远红方跨度的氦氖激光。记录微分干涉对比（DIC）的图像的非标记的组织架构进行评估。 7。代表性的成果如图ETG tTG2和IgA抗体阳性确认的一个例子。 3A。在人类患者的情况，由于DH样皮炎的发病率要低得多，比CD般的面筋敏感性也恒河猴一致，皮肤活检标本大部分预计将SHOW的tTG2抗体阳性反应，但没有tTG2 + IGA +双（图4）阳性细胞的存在。这些卫生署无关的情况下，包括微生物来源皮炎的情况下，就会发现一些单积极IGA +散落下的上皮细胞，但tTG2和IgA标记的标记（图3B和4）之间的光谱重叠。因此，重要的是要保持建议的测试程序的顺序，与临床检查（图1）病理组织学评估（图2），只有在没有其他更常见的皮炎，造成代理商可以识别，焦，开始显微镜tTG2和IgA染色的皮肤切片检查是必要的。临床和/或组织病理学评估单是不足够的阳性诊断这种情况。在这种情况下，双阳性（tTG2 + IGA +）细胞真皮乳头周围区域确定，可以发出的DH -样皮炎诊断。在这种情况下，撤回饮食面筋和管理无麸质饮食的建议，以减轻皮炎症状。 <img src="/files/ftp_upload/3154/3154fig1.jpg" alt="图1" /> 图1。EM96猕猴前腹股沟区（A）和后（b）为5个星期的饮食面筋的撤离。重新饮食面筋正好与再现性皮炎（三）。 <img src="/files/ftp_upload/3154/3154fig2.jpg" alt="图2" /> 图2 HE染色EM96猕猴的皮肤活组织切片检查，收集前（A）和（二）撤销9个星期的饮食面筋。答：上皮增厚（7-15层上皮细胞）厚的角化细胞表面的地壳变质中性粒细胞浸润。中性粒细胞也出现上皮细胞内和中等数量的淋巴细胞，浆细胞，在真皮上层的附属器周围的组织细胞混合。乙：有些hyperkerat尘埃沉着病仍然存在，上皮厚度正常化（4-7层），以最小的淋巴浆细胞浸润在真皮上部。 C：FR56猕猴腹部皮肤正常，无淋巴浆细胞浸润，包括进行比较。放大倍数20倍。 <img src="/files/ftp_upload/3154/3154fig3.jpg" alt="图3" /> 图3。EM96猕猴的皮肤活组织切片检查前收集的共聚焦显微镜图像（A）和（二）撤销9个星期的饮食面筋。答：IGA染色（绿），tTG2（红色）和TOPRO - 3核DNA标记（蓝色）。 IgA和tTG2合作定位出现在周围的皮肤papilae面积的黄色。乙：皮肤样本9周后无麸质饮食显示，上皮细胞内没有IGA，​​而一些皮下IGA仍然存在（绿色），tTG2（红色）和TOPRO - 3核DNA标记（蓝色）。 <img src="/files/ftp_upload/3154/3154fig4.jpg" alt="图4" /> 图4。 </&gt; GI24恒河猴皮肤活检共聚焦显微镜图像的收集，而动物是参展的慢性皮炎（DH -无关）症状。 IGA染色tTG2绿色，红色和TOPRO - 3核DNA标记是蓝色的。 tTG2 +细胞存在上皮细胞内，几个IGA +下方的上皮细胞，而没有广泛的光谱重叠的红色和绿色标记之间是可见的。 <table border="1"><tbody><tr><td> 动物标签 </td><td> 性别 [男/女] </td><td> 年龄 [年] </td><td> 临床症状 </td><td> 等离子tTG2 抗体 </td><td> 饮食 [1,2] * </td></tr><tr><td> HJ71 </td><td> F </td><td> 1.3 </td><td>健康</td><td> - </td><td> 1 </td></tr><tr><td> HD13 </td><td>中号</td><td> 1.4 </td><td>健康，偶有腹泻</td><td> + </td><td> 1 </td></tr><tr><td> FR56 </td><td>中号</td><td> 5.4 </td><td>慢性特发性腹泻</td><td> - </td><td> 1 </td></tr><tr><td> EM96 </td><td> F </td><td> 5.0 </td><td>慢性皮炎</td><td> + / - （边缘） </td><td> 1和2 </td></tr><tr><td> GI24 </td><td> F </td><td> 4.6 </td><td>慢性皮炎</td><td> - </td><td> 1 </td></tr></tbody></table> 表1。TNPRC恒河猴用于收集皮肤活检 *饮食1 - 定期猴子议员（含麸质）;饮食2 - 无麸质饮食

<ol>
	<li>Diosdado, B., Wijmenga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+the+adaptive,+innate+and+regulatory+immune+responses+in+the+intestinal+mucosa+of+celiac+disease+patients.">Molecular mechanisms of the adaptive, innate and regulatory immune responses in the intestinal mucosa of celiac disease patients.</a> Expert. Rev. Mol. Diagn. 5 (5), 681-700 (2005).</li><li>Schuppan, D., Dennis, M. D., Kelly, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celiac+disease:+epidemiology,+pathogenesis,+diagnosis,+and+nutritional+management.">Celiac disease: epidemiology, pathogenesis, diagnosis, and nutritional management.</a> Nutr. Clin. Care. 8 (2), 54-69 (2005).</li><li>Sardy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+transglutaminase+(TGase+3)+is+the+autoantigen+of+dermatitis+herpetiformis.">Epidermal transglutaminase (TGase 3) is the autoantigen of dermatitis herpetiformis.</a> J. Exp. Med. 195 (6), 747-757 (2002).</li><li>Rose, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+against+epidermal+transglutaminase+are+a+sensitive+diagnostic+marker+in+patients+with+dermatitis+herpetiformis+on+a+normal+or+gluten-free+diet.">Autoantibodies against epidermal transglutaminase are a sensitive diagnostic marker in patients with dermatitis herpetiformis on a normal or gluten-free diet.</a> J. Am. Acad. Dermatol. 61 (1), 39-43 (2009).</li><li>Bethune, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+transport+and+enzymatic+detoxification+of+gluten+in+gluten-sensitive+rhesus+macaques.">Transepithelial transport and enzymatic detoxification of gluten in gluten-sensitive rhesus macaques.</a> PLoS ONE. 3 (3), e1857-e1857 (2008).</li><li>Mazumdar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+transepithelial+passage+of+the+immunogenic+33-residue+peptide+from+&#x03B1;-2+gliadin+in+gluten-sensitive+macaques.">Visualization of transepithelial passage of the immunogenic 33-residue peptide from &#x03B1;-2 gliadin in gluten-sensitive macaques.</a> PLoS ONE. 5 (4), e10228-e10228 (2010).</li><li>Schuppan, D., Junker, Y., Barisani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celiac+disease:+from+pathogenesis+to+novel+therapies.">Celiac disease: from pathogenesis to novel therapies.</a> Gastroenterol. 137 (6), 1912-1933 (2009).</li><li>Hunt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+identified+genetic+risk+variants+for+celiac+disease+related+to+the+immune+response.">Newly identified genetic risk variants for celiac disease related to the immune response.</a> Nature Genet. 40 (4), 395-402 (2008).</li><li>Abadie, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+genetic+and+immunological+insights+into+a+model+of+celiac+disease+pathogenesis.">Integration of genetic and immunological insights into a model of celiac disease pathogenesis.</a> Annu. Rev. Immunol. 29, 493-525 (2011).</li></ol>

没有利益冲突的声明。

最近，我们新成立的非人类灵长类动物模型面筋敏感性是用于研究饮食面筋 5,6引发的肠道通透性变化。等离子体tTG2抗体以及肠道tTG2 + IGA +存款中检测到一些敏感的麸质的猕猴。由于我们最近认同在殖民地特发性皮炎的罕见事件恒河猴猕猴，这里没有通常的传染病/代谢的病因可能是成立，本研究的目的是，以评估如果tTG2 + IGA +抗体的存款可能被检测到的皮肤样本从这些罕见的情况下皮炎?...

<table border="1"><tbody><tr><td> 试剂或设备名称 </td><td> 公司 </td><td> 目录编号/产品编号 </td><td> 请注意 </td></tr><tr><td>抗人IGA - FITC </td><td> Sigma公司，圣路易斯，密苏里州</td><td> F - 5259 </td><td> 1:100工作液</td></tr><tr><td>反人权tTG2 </td><td> Thermo Scientific的卡拉马祖，心肌梗死</td><td> MS - 300 - P1ABX </td><td> 1:100工作液</td></tr><tr><td>抗鼠IgG1 ** </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> A - 21124 </td><td> 1:1,000工作液</td></tr><tr><td> TOPRO - 3 DNA探针*** </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> T - 3605 </td><td> 1:1,000工作液</td></tr><tr><td>锌福尔马林 （Z - FIX） </td><td> Anatech有限公司，巴特尔克里克，MI </td><td> 171 </td><td></td></tr><tr&gt; <td>华侨城冻结 中等</td><td>樱花Finetek，托伦斯，CA </td><td> 4583 </td><td></td></tr><tr><td> Cytoseal安装 中等</td><td>理查德艾伦科学，卡拉马祖，心肌梗死</td><td> 8312-4 </td><td></td></tr><tr><td>反淬火 解决方案</td><td> Sigma公司，圣路易斯，密苏里州</td><td> P 3130 </td><td></td></tr><tr><td> Ethilon （尼龙缝线） </td><td>爱惜康公司，强生公司，英国伯克郡， </td><td> 663G </td><td></td></tr><tr><td> Miltex打孔真皮活检仪器</td><td> Miltex，纽约，PA </td><td>不适用</td><td></td></tr><tr><td>奥斯特金A5单速度与规模的40刀片兽医快船</td><td>奥斯特产品，麦克明维尔，TN </td><td> 78005-050 </td><td></td></tr><tr><td>徕卡组织处理器</td><td>莱卡微系统公司布法罗树丛，KS </td><td> ASP300 </td><td></td></tr><tr><td>旋转式切片机</td><td> MICROM国际 华尔道夫，德国</td><td> HM325 </td><td></td></tr><tr><td>低温恒温器</td><td> Thermo Scientific的， 卡拉马祖MI </td><td> HM560 </td><td></td></tr><tr><td>共聚焦显微镜</td><td>莱卡，韦茨拉尔 德国</td><td> TCS SP2 </td><td></td></tr></tbody></table> *异硫氰酸荧光素 **中使用抗体后tTG2初级抗体（共轭的Alexa Fluor 568） **染料

recognition of epidermal transglutaminase by iga and tissue transglutaminase 2 antibodies in a rare case of rhesus dermatitis

组织转谷氨酰胺酶2（tTG2）是肠道的消化酶deamidates已经部分消化的饮食面筋如醇溶蛋白肽。在遗传倾向的个体，tTG2触发自身免疫反应的特点是由生产tTG2抗体和直接沉积到小肠壁 1,2 。这种抗体的存在，构成腹腔病（CD）的重要标志之一。表皮转谷氨酰胺酶（ETG），也可以作为在少数的CD患者的自身抗原的转谷氨酰胺酶家族的另一成员。在这些比较少见的情况下，ETG触发自身免疫反应（皮疹），临床上称为疱疹性皮炎（DH）。虽然CD和卫生署的发病的确切机制还不是很清楚，它是已知tTG2和血清抗体可以识别CD和卫生署病人 3,4 ETG份额抗原表位。 猕猴 ）皮炎（表1，图1和2）皮损活检标本用于研究受影响的组织。在一个动物（EM96）IgA和tTG2抗体（图3）光谱重叠证明。集中在深的表皮，真皮​​乳头周围，存在的双阳性tTG2 + IGA +细胞。这是符合卫生署病人描述的病变 3 。当EM96是一个无麸质饮食，皮炎，以及放置tTG2 + IGA +存款消失，不再检测到（图1-3）皮炎再现然而，基于EM96的饮食面筋（未显示）的重新引入。包括动物无关皮炎的其他猕猴，tTG2 + IGA +存款均未检出。无麸质饮食依赖性皮炎缓解EM96 tTG2存在+ IGA +在它的皮肤细胞表明自身免疫性疾病，卫生署状这种情况的发展机制。这是任何非人类灵长类动物中的DH -样皮炎的第一次报告。

Watch this Scientific Journal Video about Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis at JoVE.com

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

疱疹性皮炎（DH）是一种慢性炎症性IGA和表皮转谷氨酰胺酶（ETG）之间的自身免疫反应的条件。卫生署发展中的麸质敏感和/或腹腔的患者非常小的部分。这项研究的结果表明，卫生署也可以在idiopatic皮炎症状猕猴主机发展。

在极少数情况下，表皮转谷氨酰胺酶IgA和组织转谷氨酰胺酶2抗体的识别恒河猴皮炎

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

recognition-epidermal-transglutaminase-iga-tissue-transglutaminase-2

Research

JoVE Journal

Immunology and Infection

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

24.4K 次观看.  Tulane National Primate Research Center. 疱疹性皮炎（DH）是一种慢性炎症性IGA和表皮转谷氨酰胺酶（ETG）之间的自身免疫反应的条件。卫生署发展中的麸质敏感和/或腹腔的患者非常小的部分。这项研究的结果表明，卫生署也可以在idiopatic皮炎症状猕猴主机发展。

视频: Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

多色流流式细胞仪分析细胞在恒河猴的免疫应答

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

科研

免疫与感染

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IgY的技术：从蛋黄鸡抗体提取聚乙二醇（PEG）降水量

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

Antibody Binding Specificity for Kappa (V&amp;#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

附NCAM作为一个案例研究聚唾液酸（PSA）：为河童（Vκ）抗体结合特异性含轻链人类（IgM抗体）抗体

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

鼠单克隆抗体的产生杂交瘤技术

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

猴免疫缺陷病毒特异性CD8分析<sup&gt; +</sup&gt; T细胞猕猴由肽MHC-I四聚体染色

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

甲醛暴露对大鼠新生辣椒素致过敏性皮炎模型的影响

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...