南希·斯特姆，免疫学，微生物学和分子生物学，大卫格芬医学院，加州大学洛杉矶分校，批判性阅读的手稿，我们非常感激。国民议会研究CNPq的，基础研究发展FAPDF，巴西，支持本研究。我们感谢澳亚历山德罗·索萨，玛丽亚三Guimaro，西罗郭利民，安娜·卡西亚·罗萨，Roseneide阿尔维斯，和拉斐尔·安德拉德的技术援助，从巴西，巴西利亚大学。

1。生长寄生虫<ol><li> T 的成长trypomastigote形式锥虫 Berenice和β-半乳糖苷酶表达的Tulahuen 吨锥虫 MHOM/CH/00的C4（L6）在小鼠肌肉细胞培养杜尔贝科最小的重要媒介与10％的FSB，100 IU / ml青霉素，100微克/毫升链霉素，和250 nm的L -谷氨酰胺（pH值7.2），5％ CO 2在37°C。使用中上清培养基自由游泳trypomastigotes，，被接种鸡蛋。 </li><li>成长， 利什曼braziliensis（LB）在DMEM LTB300股票增长20％胎牛血清。在的LB promastigote形式的指数增长阶段，用于接种鸡蛋9。 </li></ol> 2。寄生虫接种在受精鸡蛋<ol><li>接种了100 吨，取消冻结锥虫 trypomastigotes在鸡蛋壳上的阶段顶部的气室，通过一个直径为2毫米的孔10μL培养基X肥沃的鸡蛋。进入胚胎细胞的致命寄生虫的侵袭和复制显示在视频S1。对照组如下：a）控制鸡;二）接收模拟控制鸡蛋10μL培养基; C）X期受精卵与暂停100 磅 promastigotes的接种在培养基中加入10μl 吨 。 锥虫和LB属于的kinetoplastid家庭。分别，这些原生动物生长在细胞质中，或在宿主细胞的虫空泡10。 </li><li>用胶带封洞。 </li><li>孵育的T。克氏锥虫感染的鸡蛋模拟和未感染的对照样品在37.5°C和65％，湿度为21天。 </li><li>三个星期的小鸡，在孵化器中孵化24小时，其后保持在32°C。 </li></ol> 3。获取样本DNA的提取<ol><li>鸡外周血单个核细胞获得一）孵化从T.接种锥虫鸡蛋;二）管制; C）嘲笑接收10微升培养基，D）从LB接种蛋孵化鸡白血细胞DNA的提取与处理标准协议11。 </li><li>还从公鸡，母鸡孵鸡蛋与T.接种收集贫瘠的卵母细胞（&lt;5毫米）收集精液中提取DNA 克氏锥虫 ，并从控制鸡蛋3个，4孵出母鸡。 </li><li>提取的T. kDNA 锥虫 epimastigote形成，也从LB promastigotes，所述其他地方9。 </li></ol> 4。所用引物和探针引物用于PCR扩增和热条件如表1所示。 在Southern印迹杂交用的探针分别为： <ol><li>野生，类型minicircle（〜1.4 KB）序列纯化田间从T.锥虫 epimastigote形式; </li><li> Minicircle片段（362 bp）的由NSI取得的我的野生型kDNA消化; </li><li>核DNA（nDNA的）重复序列（188 bp）的寄生虫DNA扩增获得与Tcz1 / 2的引物。探针进行纯化，从1％琼脂糖凝胶3。 </li><li>野生，类型minicircle（〜0.820 KB）序列磅 promastigotes。 </li></ol> 5。聚合酶链反应分析<ol><li>基因组DNA从被感染的雏鸡和感染的控制与运行标准PCR程序和嘲笑使用吨锥虫 nDNA的Tcz1 / 2 12和kDNA s35/s36的13引物。此外，运行磅，使用的原生动物的具体LB3和LB5引物（ 表1）感染的鸡蛋孵化鸡的基因组的DNA PCR。 </li><li> 100 ng的模板DNA，每对引物，2个U Taq DNA聚合酶，0.2毫米的dNTP，0.4微米和1.5毫米氯化镁，使反应混合物<sUB&gt; 25微升的最终体积。 </li><li>在68°，在72分钟的C / 1°C间前5分钟内制冷最后一次延长为5分钟，30秒30的周期在95°C/30的秒热循环程序为95℃。 </li><li>扩增产物在1.3％琼脂糖凝胶转移，这是由一个带正电的尼龙膜（GE生命科学）的碱性标记的特异性探针杂交方法分析[α-32 P] dATP的使用随机引物标记试剂盒（Invitrogen公司卡尔斯巴德，加利福尼亚州）。 </li></ol> 6。基因组Southern杂交<ol><li>使用MBO我和/或生态的RI（Invitrogen）的酶，使单一集成到人体组织的DNA样本minicircles削减。 </li><li>从感染的控制鸡和鸡的精华DNA孵化蛋从剧毒吨接种锥虫形成。 </li><li>主题摘要从 T的DNA 克氏锥虫和从鸡测试样品在0.8％琼脂糖凝胶电泳，在50 V过夜4°C </li><li>将分离的DNA带正电的尼龙膜。 </li><li>无线电标记kDNA探针杂交的DNA条带。 </li><li>洗膜两次15分钟65°2X SSC和0.1％SDS，两次15分钟65°C间0.2X SSC和0.1％SDS，时间变量的放射自显影。 </li></ol> 7。有针对性的总理TAIL-PCR技术<ol><li>获取修改后的TAIL-PCR技术的技术，它结合了kDNA特异引物引物2 minicircle整合到鸡的基因组扩增的kDNA三个走在巢式PCR的周期， 如图1所示。 </li><li>主要周期：每个反应包括200 ng的模板DNA，MgCl 2的 2.5毫米，0.4μM的，kDNA引物（S34或S67），0.2毫米dNTPs浓度，2.5 U的Taq铂（Invitrogen公司，卡尔斯巴德，CA）使用GG的引物（GG1 Gg6， 表1）的0.04微米kDNA引物组合，分别。设定温度从57.9到60.1°kDNA引物和从59.9至65.6℃，CR-1引物，请注意，这些温度高于（〜45°）退化的任意引物TAIL-PCR技术10。使用温度和周期（MyCycle热循环仪，Bio-Rad公司实验室，大力士，CA）在以前的文件3所述。 </li><li>第二周期：从主要周期1:40（V / V）水稀释PCR产物。 kDNA引物S35和S35反义取代以往，伴随着同样的GG引物。 </li><li>第三周期：从二次周期1:10（V / V）水稀释PCR产物，并结合千吨引物，分别与S67反义或S36。 </li><li>克隆的PCR第三周期的产品：直接克隆至pGEM T easy载体（Promega公司，麦迪逊，威斯康星州）上放大产品kDNA探针杂交。 </li><li>选择，kDNA探头和序列杂交的克隆。 </li><li>从T.在300 kDNA PG混合验证tpTAIL-PCR 锥虫控制鸟类的DNA与200纳克从未暴露kDNA。用于测试鸟类DNA相同的温度和放大周期。 </li></ol> 8。南美锥虫病的临床表现<ol><li>监测的增长和发展，从T.孵化鸡锥虫感染的鸡蛋来自非疫区的鸡蛋孵化和健康对照组的死亡率和疾病表现每周每天。 </li><li>检测这些鸡的临床异常（ 图2）和心电图（ECG）记录，以评估电轴，心脏率和心律失常3。 </li><li>主题kDNA突变和控制鸡每月向增强心室联合国的心电图记录的iPolar导致aVF导联（左腿），aVL导联（左臂），AVR（右臂），并评估偏差的平均电轴左，这是提示心脏扩大3。 </li></ol> 9。病理和免疫组化分析<ol><li>自然kDNA突变鸡死亡后记录心脏和体重指数（ 图3）。获得指数也相同年龄和性别控制鸡。 </li><li>采取心脏，食道，肠，骨骼肌，肺，肝，肾脏的部分。 </li><li>修复组织中10％的福尔马林缓冲液（pH 7.4），石蜡嵌入和削减至4微米厚切片苏木素-伊红（HE）染色和病理分析（ 图3）。 </li><li>从胚胎的收获和对开组织接种表达β-半乳糖苷酶的寄生虫卵，孵化，X-Gal的染色。 </li><li> 1修复的胚胎组织中的另一半0％福尔马林，pH值7.4，并继续在步骤9.3。 </li><li>切4μm的薄石蜡包埋组织切片，并安装在玻璃镜检查幻灯片。 </li><li>孵育部分显示X-Gal的蓝染细胞与人类的查加斯氏抗血清（1:1024稀释）反对抗-T。克氏锥虫抗原。 </li><li>与PBS，pH值7.4，5帕特里斯分钟每洗节。 </li><li>染色蓝色细胞在胚胎组织，通过与荧光标记的兔抗人IgG第二孵化。 </li><li> （步骤8）用PBS洗节，安装盖玻片，观察光，紫外光照射下，蓝色细胞绿色经审查，在502 nm波长，200X放大倍率，同时位吨 ， 锥虫在胚胎细胞。 </li></ol> 10。在心脏病变型免疫系统的细胞<ol><li>型免疫效应细胞在心脏组织切片从kDNA阳性和控制kDNA负鸡。 </li><li>将幻灯片组织切片，石蜡包埋，65°C时为30分钟，每次5分钟，融化在100％至70％的二甲苯，然后在无水乙醇PBS蜡4冲洗以前提交。 </li><li>幻灯片冲洗，蒸馏水，空气干燥，以及治疗特异性单克隆抗体获得从SouthernBiotech，伯明翰，AL（荧光的R-藻红蛋白标记的单克隆抗体）。 <ol><li>用鼠标抗鸡BU-1（BU-1 a和BU-1 B等位基因，先生70-75 kDa的）单克隆抗体AV20承认B细胞抗原对自交系鸡的单形性的决定因素。 </li><li>用鼠标抗鸡，CD45，免疫球蛋白亚型IgM1κ特定鸡胸腺细胞系（先生190到215 kDa的变种）。 </li><li>使用鼠抗鸡TCRγδ+（90-kDa的异先生）单克隆抗体的具体胸腺依赖CD8α+ T细胞。 </li><li>使用鼠标抗鸡抗体CD-8鸡α链（议员34 kDa的）承认日ËCD8细胞在胸腺，脾脏，心脏和其他组织。 </li><li>使用鼠标抗鸡KuL01的专门识别单核/巨噬细胞的吞噬细胞系统。 </li></ol></li><li>洗三次幻灯片用0.1 M PBS，pH值7.4，5分钟内每一个具体的反型抗体在潮湿室90分钟后孵化。 </li><li>组装与缓冲考试甘油，荧光灯显微镜下用波长567和502 nm的发射滤波器的幻灯片，分别检测红色和绿色荧光标记的细胞（ 图4）。 </li></ol> 11。数据分析<ol><li>使用鸡基因组数据库（ <a href="http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=9031">http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=9031的</a> BLASTN序列分析）。 </li><li>使用CLUSTALW路线确定E值的分数。 </li><li>聘请的GI国际扶轮重复屏蔽算法御史（ <a href="http://girinst.org/censor/index.php">http://girinst.org/censor/index.php</a> ）的不同类别的嵌合序列重复定位。 </li><li>聘请Kinetoplastid插入和删除序列搜索工具（吻）kDNA序列潜在gRNAs与WU-BLASTN改良基质3的援助，以确定。 </li><li>使用T。锥虫序列<a href="http://www.biomedcentral.com/content/supplementary/1471- 2164-8-133-s1.fas">http://www.biomedcentral.com/content/supplementary/1471- 2164-8-133-s1.fas</a>搜索，在kDNA宿主DNA嵌合体gRNAs。 11.6）使用学生的牛逼和“Kolmorov-斯米尔诺夫测试，分别为，以检测与电动轴的偏差和心脏/身体体重指数之间的显著实验组和对照组获得的差异，并以检测死亡率比孵鸡的群体之间的显著差异从T.锥虫我noculated鸡蛋和控制。 </li></ol> 12。代表结果接种100剧毒吨的到的锥虫 trypomastigotes不肥沃的鸡蛋气室不显着减少孵化活着的小鸡比例。约60％，孵出的健康雏鸡和40％可能发生在孵化的胚胎液化或胚胎死亡。尚存的雏鸡保留的kDNA minicircle序列基因组中的集成。但是，它预计，一些雏鸡孵化后会死在几周内与心脏和失败。其余的雏鸡将增长到外表健康的成年人。在人生的所有阶段，从他们的血液细胞中提取的DNA将产生kDNA PCR扩增，但不nDNA的。目标首相尾-PCR扩增3，4个产品被克隆和序列将显示，kDNA minicircles主要集成在编码macrochromosomes地区1至5。显示多个kDNA我的鸡进入细胞的生长和分化，调节免疫系统的因素，和DNA修复基因编码的ntegrations是候选人进行自我靶组织（ 图3）的拒绝。例如，鸡与dystrophin基因的断裂（ 图5）显示kDNA突变，编码的蛋白质结合到细胞膜的骨架，是一个以发展自身免疫性炎症性心肌病和失败的候选人。 这些基因的修改都没有见过在磅感染的鸡蛋孵出的小鸡。 吨之间有分歧克氏锥虫和LB kDNA的minicircles;的T.穿插锥虫ķDNA minicircle平均1.4 kb的四个可变区（VR）的结构保守的区域，每个提出CSB1（CR）CSB2，和CSB3区域，其中被认为是CA丰富的弯曲的DNA复制起始的具体地点，转录，重组，横向基因转移<s高达4&gt; 3。 磅，，kDNA minicircle（平均大小820 bp）的，截然不同的包含单CR自愿退休。 CR已保守CSB1（GGGCGT）和CSB2（CCCCGTTC）块，这是不同于吨锥虫 minicircles 15，16和17。考虑，LB CSB3（GGGGTTGGTGTA）显示12 NTS同源的T。克氏锥虫 ，可以想象，无论是LB kDNA minicircle集成在一个多低的频率，这可能不是所使用的技术，可见，或可能，可能，而不是在鸡基因组整合。 <table border="1"><tbody><tr><td> 底漆 </td><td> 目标DNA </td><td> 序列 </td><td> TM * </td></tr><tr><td>第34号</td><td> T.锥虫 kDNA </td><td> 5&#39;ACA的行政协调会文建会文建航管棉酚的CC 3&#39; </td><td> 57.9 </td></tr><tr><td>第67号</td><td> T。锥虫kDNA </td><td> 5&#39;GGT的TTT GGG AGG的GG（的G / C）（G / C）（T / G）TC 3&#39; </td><td> 60.1 </td></tr><tr><td> S 35 </td><td> T.锥虫 kDNA </td><td> 5&#39;ATA ATG的交咨会GGG（的T / G）遗传算法的GAT GC 3&#39; </td><td> 59.4 </td></tr><tr><td> S 36 </td><td> T.锥虫 kDNA </td><td> 5&#39;GGT的TCG的ATT GGG滋养细胞GGT的G 1“ </td><td> 57,9 </td></tr><tr><td> LB3 </td><td> 磅 kDNA </td><td> 5&#39;GGG GGT的糖耐量多伦多的ATA豪TGG G 1“ </td><td> 55.9 </td></tr><tr><td> LB5 </td><td> 磅 kDNA </td><td> 5&#39;CTA ATT GTG CAC GGG GAG的G 1“ </td><td> 61.4 </td></tr><tr><td> GG 1 </td><td> 鸡内金 </td><td> 5&#39;AGC TGA台泥的TAA AGG的造影AGC 3&#39; </td><td> 60.1 </td></tr><tr><td> GG2 </td><td> G.的鸡 </td><td> 5&#39;CTG的AGC的反恐TGCTTT棉酚3&#39; </td><td> 56.8 </td></tr><tr><td> gg3 </td><td> G.的鸡 </td><td> 5&#39;TTT民航局AGC AGA GGC TCG的G 1“ </td><td> 60.1 </td></tr><tr><td> gg4 </td><td> G.的鸡 </td><td> 3，GCT公司CTG的建TTA GGA TCA的GCT的5&#39; </td><td> 64.2 </td></tr><tr><td> gg5 </td><td> G.的鸡 </td><td> 3&#39;AGC的AAC TCA的图文影像集团台泥行政协调会测控5&#39; </td><td> 62.3 </td></tr><tr><td> gg6 </td><td> G.的鸡 </td><td> 3&#39;CTG的TTA GCA的热重GGC TTC ACA的5&#39; </td><td> 60.4 </td></tr></tbody></table> 表1。素数用于PCR扩增。 * TM =平均退火温度°C。 <a href="http://www.jove.com/files/ftp_upload/3716/3716fig1_lg.jpg"><img src="/files/ftp_upload/3716/3716fig1.jpg" alt="图1" /></a> 图1。TP TAIL-PCR技术战略用来检测到鸡内金基因组克氏锥虫 kDNA整合。 A）与kDNA片段嵌合序列保守（深蓝色）和可变minicircle（淡蓝色）在4号染色体的10鸡的基因组（绿色）（AY237306）在轨迹NW_001471687.1集成的区域被用来获取主机特异引物集（GG1 Gg6）。乙）的TP TAIL-PCR技术扩增发起的minicircle特定的S34或S67引物组合鸡具体的GG1到Gg6引物退火（主循环）。稀释后的产品提供二级周期的S35（正义/反义）引物和GG 的引物组合的模板。在第三周期，二级品稀释遭到kDNA S36或S67反义引物扩增与GG 的引物组合第c）这些扩增产物进行分离，在1％琼脂糖凝胶转移到尼龙膜上，与具体的kDNA探针杂交。样品呈现出积极的信号被用于克隆确定的结合点。凝胶上显示的组合kDNA GG1到Gg6针对性。连续PCR反应扩增kDNA minicircles（蓝色）和禽流序列（绿色）的目标kDNA宿主的DNA序列。 （3 公共科学图书馆被忽视的热带病重印）。 <img src="/files/ftp_upload/3716/3716fig2.jpg" alt="图2" /> 图2。基因线粒体kDNA的集成修改了9个月大的鸡的心脏功能受损的临床表现minicircle从T.锥虫 。血氧差的线粒体kDNA突变紫色的梳子对比显示与控制9个月大的chicke明亮的红色的鸡冠鸡列印无心脏损害。 （3 公共科学图书馆被忽视的热带病的修改）。 <img src="/files/ftp_upload/3716/3716fig3.jpg" alt="图3" /> 图3。总值和微观病理kDNA突变与棒棒鸡 。一）心脏在9个月的老母鸡，心脏衰竭死亡。 b）从一个未感染的9个月的老母鸡控制心脏。 ）目标的心脏细胞的细胞毒性T细胞的抑制：描绘与最小抑制免疫淋巴细胞对靶细胞裂解酶单位（圆圈）。 D）控制心脏组织学（3 公共科学图书馆被忽视的热带病的修改）。 <img src="/files/ftp_upload/3716/3716fig4.jpg" alt="图4" /> 图4。浸润心脏如图3所示的kDNA突变鸡的免疫系统细胞的免疫细胞化学分析。一）CD45的+淋巴细胞在心脏LE（箭头）sions由1藻红蛋白标记的特异性单克隆抗体。二）CD8 + +γδ免疫的淋巴细胞（箭头）参与在心脏的严重破坏。三）丰富的CD8α+ T细胞中存在严重的病变心脏细胞裂解酶。插入显示在控制感染的鸡的心（3 公共科学图书馆被忽视的热带病的修改）的情况下对免疫系统的细胞。 <img src="/files/ftp_upload/3716/3716fig5.jpg" alt="图5" /> 图5。恰加斯样扩张炎症性心肌病在kDNA在dystrophin基因的整合的F2代后代。一）扩张型心肌病心脏占据大部分的胸腔（心脏重量=16克），在10个月大的鸡。 b）暗轮单核细胞浸润和破坏心肌kDNA突变的母鸡。 c）10个月大的控制鸡的正常心脏的大小（重量7克）。四）控制鸡心脏的正常组织学。 （从公共科学图书馆被忽视的热带修改人疾病3）。 <img src="/files/ftp_upload/3716/3716fig6.jpg" alt="图6" /> 图6。比较kDNA变异鸡和人类南美锥虫病的病理。 a）严重心肌炎和目标的心脏细胞裂解酶在鸡kDNA突变。 b）严重心肌炎和靶细胞裂解酶的的恰加斯心脏疾病的情况下，免疫淋巴细胞。三）拒绝心脏细胞在kDNA突变的鸡的免疫淋巴细胞。 d）抑制心脏细胞在人类南美锥虫病的免疫淋巴细胞。 Hematoxilin和曙红染色。 （来自Memórias的修改做研究所奥斯瓦尔多·克鲁斯 ，里约热内卢14）。

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIK+and+IKKbeta+interdependence+in+NF-kappaB+signalling--flux+analysis+of+regulation+through+metabolites.">NIK and IKKbeta interdependence in NF-kappaB signalling--flux analysis of regulation through metabolites.</a> Biosystems. 99, 140-149 (2010).</li><li>Karakhanova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK/p38+MAP-kinases+and+PI3K+are+involved+in+the+differential+regulation+of+B7-H1+expression+in+DC+subsets.">ERK/p38 MAP-kinases and PI3K are involved in the differential regulation of B7-H1 expression in DC subsets.</a> Eur. J. Immunol. 40, 254-266 (2010).</li><li>Finsterer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heart+in+human+dystrophinopathies.">The heart in human dystrophinopathies.</a> Cardiology. 99, 1-19 (2003).</li><li>Nitz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+integration+of+kDNA+minicircle+sequences+from+Trypanosoma+cruzi+into+the+avian+genome:+insights+into+human+Chagas+disease.">Heritable integration of kDNA minicircle sequences from Trypanosoma cruzi into the avian genome: insights into human Chagas disease.</a> Cell. 118, 175-186 (2004).</li><li>Simpson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetoplast+DNA+in+trypanosomid+flagellates.">Kinetoplast DNA in trypanosomid flagellates.</a> Int. Rev. Cytol. 99, 1-19 (1986).</li><li>Bonney, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-killed+Trypanosoma+cruzi+induces+acute+cardiac+damage+and+polyantigenic+autoimmunity.">Heat-killed Trypanosoma cruzi induces acute cardiac damage and polyantigenic autoimmunity.</a> PLoS One. 6, e14571 (2011).</li><li>Moser, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Trypanosoma+cruzi+by+DNA+amplification+using+the+polymerase+chain+reaction.">Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction.</a> J. Clin. Microbiol. 27, 1477-1482 (1989).</li><li>Sturm, 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=Sensitive+detection+and+schizodeme+classification+of+Trypanosoma+cruzi+cells+by+amplification+of+kinetoplast+minicircle+DNA+sequences:+use+in+diagnosis+of+Chagas'+disease.">Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas' disease.</a> Mol. Biochem. Parasitol. 33, 205-214 (1989).</li><li>Teixeira, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+pathology+in+chagas+disease--a+review.">Evolution and pathology in chagas disease--a review.</a> Mem. Inst. Oswaldo Cruz. 101, 463-491 (2006).</li><li>Yurchenko, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Leishmania+minicircle+kinetoplast+DNA+classes.">Structure of Leishmania minicircle kinetoplast DNA classes.</a> J. Clin. Microbiol. 37, 1656-1657 (1999).</li><li>Simpson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+organization+of+guide+RNA+genes+in+kinetoplastid+protozoa:+several+conundrums+and+their+solutions.">The genomic organization of guide RNA genes in kinetoplastid protozoa: several conundrums and their solutions.</a> Mol. Biochem. Parasitol. 86, 133-141 (1997).</li><li>Thomas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-universal+transcription+factor?+The+Leishmania+tarentolae+TATA+box-binding+protein+LtTBP+associates+with+a+subset+of+promoters.">A non-universal transcription factor? The Leishmania tarentolae TATA box-binding protein LtTBP associates with a subset of promoters.</a> Int J. Parasitol. 36, 1217-1226 (2006).</li></ol>

没有利益冲突的声明。

相反到哺乳动物容易受到终身吨克氏锥虫感染，鸡难治吨克氏锥虫感染。鸡模型系统的主要优点是消除感染的早期胚胎的免疫系统的发展。因此，唯一的寄生虫在鸡体内剩余的DNA被整合在几个位点。 使用剧毒吨的最优数量锥虫 trypomastigotes接种受精卵是向鸡胚胎的基因组整合kDNA minicircles获得关键的一步。现场的小鸡从100 trypomastigotes接种的鸡蛋孵化的速度是比获得500寄生虫的高4倍。应小心在10μL培养基接种到鸡蛋的气室中的寄生虫暂停。应该是没有蛋清泄漏。在最佳条件下，细胞内的寄生虫感染发生无线此后的感染是由先天免疫淘汰;几个小时后，一个星期内的宿主细胞所得的孵化和寄生虫乘法薄。 kDNA整合需要一个生活的感染，并不会导致融合成早期胚胎鸡蛋裸minicircles接种。在受控条件下的的kDNA阳性胚胎和控制应该被安置在37.5°C和65％的湿度。雏鸡在33℃室温存放在两个星期的笼子。此后，鸡在笼子里的暂停架1.5米宽的过道隔开的房间在22°C间过滤空气和积极的压力，以确保动物福利条件下不断枯竭。成人喂养的鸡议员，喝饮用水的自来水，实现全面成长和成熟，铺设鸡蛋在5个月的年龄。卫生程序维护结果的重复性的基本以 T工作时锥虫接种成肥沃的鸡蛋。 在鸡模型系统的T.克氏锥虫感染根除后，免疫系统的发育在胚胎发育的初期。此外被感染，从T.孵化小鸡锥虫接种的鸡蛋，在缺乏特异性抗体，是宽容的寄生虫抗原。 kDNA突变基因型修改和免疫监视击穿3鸡只被视为拒绝目标的心脏细胞的细胞毒性T细胞（最小单位拒绝， 图3）。基因型修改的T细胞呈现在体内自我组织加速排斥反应。主要病变部位是心脏，这是南美锥虫病的一个标志。从生理（监察）通过一个病理生理状态，显示克隆3细胞毒性T细胞增殖的在kDNA突变的鸡。 ţ他transkingdom模型系统显示了一种寄生虫引起的，基因驱动的自身免疫性疾病（ 图6），从基因组的修改所产生的T。锥虫 kDNA minicircle集成。这些修改都没有见过从磅接种的鸡蛋孵出小鸡。 这种现象表明，炎症在kDNA突变鸡的自身免疫性心肌病的实验性治疗，可能需要药物抑制骨髓祖细胞浸润心肌特异性T细胞型，histocompatible健康的骨髓移植，以防止排斥反应的自我组织。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>的Taq DNA聚合酶重组</td><td> Invitrogen公司</td><td> 11615-010 </td><td></td></tr><tr><td>铂金的Taq DNA聚合酶</td><td> Invitrogen公司</td><td> 10966-030 </td><td></td></tr><tr><td>随机引物DNA标记系统</td><td> Invitrogen公司</td><td> 18187-013 </td><td></td></tr><tr><td> 生态国际扶轮</td><td> Invitrogen公司</td><td> 15202-021 </td><td></td></tr><tr><td> MBO我</td><td> Invitrogen公司</td><td> 15248-016 </td><td></td></tr><tr><td>核苷酸集，100mm的解决方案</td><tD&gt; GE医疗</td><td> 28-4065-51 </td><td></td></tr><tr><td> Amersham公司Hybond - N + -猫列印。 </td><td> GE医疗集团</td><td> RPN303B </td><td></td></tr><tr><td> PlasmidPrep迷你自旋套件</td><td> GE医疗集团</td><td> 28-9042-70 </td><td></td></tr><tr><td> NSI我</td><td> Sigma-Aldrich公司</td><td> R5884每千</td><td></td></tr><tr><td> DNA，钠盐鱼精子</td><td> AMRESCO </td><td> 0644-10G </td><td></td></tr><tr><td>鼠抗鸡卜-1B </td><td> SouthernBiotech </td><td> 8370-02 </td><td></td></tr><tr><td>鼠抗CD45的鸡</td><td> SouthernBiotech </td><td> 8270-08 </td><td></td></tr><tr><td>鼠抗鸡TCRγδ </td><td> SouthernBiotech </td><td> 8230-08 </td><td></td></tr><tr><td>鼠抗鸡CD8α </td><td> SouthernBiotech </td><td> 9220-02 </td><td></td></tr><tr><td>鼠抗鸡单核细胞/巨噬细胞</td><td> SouthernBiotech </td><td> 8420-02 </td><td></td></tr><tr><td> MyCycle Termocycler </td><td> BIO-RAD实验室</td><td> 580BR 5501 </td><td></td></tr></tbody></table>

parasite induced genetically driven autoimmune chagas heart disease in the chicken model

克氏锥虫在婴幼儿急性感染收购似乎无症状，但约三分之一的慢性感染的个案表明达三十年或稍后的南美锥虫病。自身免疫性疾病和寄生虫的持久性竞争理论来解释1，2南美锥虫病的发病机制。南美锥虫病寄生虫的持久性和自身免疫性疾病中发挥不同的角色，我们接种的T。锥虫在气室的受精卵。成熟的鸡的免疫系统是一个严密的对T的生物屏障克氏锥虫感染根除的第一个星期增长3月底后，其免疫系统的发展。在孵化雏鸡寄生虫，但他们保留集成寄生虫线粒体动基体DNA（kDNA）minicircle在他们的基因转移到他们的后代。 kDNA在鸡基因组minicircle集成的文档由TARG获得eted总理TAIL-PCR技术，南方杂交，克隆和测序3，4。的kDNA minicircle集成破裂转录和免疫系统因素的开放阅读框，磷酸酶（GTP酶），腺苷酸环化酶和磷酸化酶（PKC，NF-κB的激活，PI-3K）与细胞的生理，生长，分化3，5 - 7，其他基因的功能。重症心肌炎，因拒绝目标心脏纤维细胞毒性T细胞效应被认为是在的kDNA突变鸡，表现出类似人类南美锥虫病看出，炎症性心肌病。值得注意的是，心脏衰竭及骨骼肌无力成年鸡目前在1号染色体8 kDNA dystrophin基因的断裂。类似genotipic改变与CD45的效应+，CD8γδ的，CD8α淋巴细胞进行组织破坏。因此，这种原虫感染可诱发基因驱动的自身免疫性疾病。

Watch this Scientific Journal Video about Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model at JoVE.com

Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model

接种<em&gt;克氏锥虫</em&gt;在之前孵化的受精卵呈现寄生虫kDNA minicircle胚胎细胞的基因组中的整合。杂交揭示突变的垂直转移到后代。该的kDNA集成到编码在几个染色体区域和鸡只死亡与自身免疫性炎症心脏疾病。

寄生在鸡模型诱导自身免疫性基因驱动恰加斯心脏疾病

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases ...

parasite-induced-genetically-driven-autoimmune-chagas-heart-disease

Research

JoVE Journal

Immunology and Infection

13.7K 次观看.  University of Brasilia. 接种克氏锥虫在之前孵化的受精卵呈现寄生虫kDNA minicircle胚胎细胞的基因组中的整合。杂交揭示突变的垂直转移到后代。该的kDNA集成到编码在几个染色体区域和鸡只死亡与自身免疫性炎症心脏疾病。

视频: Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

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

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

SJL小鼠诱导实验性自身免疫性垂体炎

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

科研

免疫与感染

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.

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

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

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

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

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

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

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

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a&#160;chagasic mother to her offspring, but the male partner&#39;s contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody.&#160;Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.

The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.

美国锥虫从男性和女性到天真的命运的性传播

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

登革热病毒诱发急性病毒性脑炎样疾病的鼠害模型

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

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

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

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

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

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

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

在伊蚊中传播微孢子寄生虫Edhazardia aedis

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

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.