We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

在美国地质调查局国家野生动物健康中心进行的动物工作是按照实验动物福利准则NIH办公室和体制下的动物护理和使用委员会的协议＃EP080716执行。从狩猎采集动物组织来自威斯康星州或自然资源密歇根部门礼物。 1.溶液制备注意:下面列出的解决方案都需要准备以下第2的CER检测基材。制备各从较高浓度的储备溶液，这些解决方案;推荐浓度原液将括号中所列各有关化学名后给出。准备好所有的解决方案在基板制备和储存新鲜的在4°C，直到使用。 <ol><li>为裂解缓冲液，制备下列在10mM Tris，pH值7.5（的1M Tris，pH值7.5原液推荐）中的溶液:100mM的s裂果氯化物（氯化钠，1M储备溶液），10毫乙二胺四乙酸（EDTA，500毫摩尔EDTA，pH 8.0的储备溶液），0.5％NP-40（10％储备液），0.5％脱氧胆酸盐（DOC，10％原液）。 </li><li>对转换缓冲，制备0.05％的十二烷基硫酸钠（SDS，10％储备液）和0.5％的Triton X-100（10％储备液）在1磷酸盐缓冲盐水（PBS，10，pH 7.4的储备液）的溶液中。 </li><li>对于离液的解决方案，准备盐酸胍（盐酸胍，8M的股票）在1X PBS（10X原液）两个3米的解决方案。用浓盐酸（HCl）调节的解决方案之一的pH至3.5±0.05。检查第二溶液的pH值保持在pH 7.4±0.05和调整用浓盐酸或氢氧化钠（NaOH）中，如果必要的。 </li></ol> 2.准备CER检测底物双<ol><li>获得健康的，非TSE感染脑组织从感兴趣的物种和制备10％重量每体积（重量/体积）匀浆中使用以下任何一种方法的裂解缓冲液:DOUNCE，珠磨机或研钵和杵均化。进一步细化导致匀浆对他们进行注射器和针头的同质化，利用增加计针，直到基材可以被吸入并通过27G的注射器针头排出自如。 <ol><li>从啮齿动物和其他小型哺乳动物准备基板，均质全脑;（二）从大型哺乳动物准备基板，使用OBEX（脑干）组织准备匀浆。当选择脑匀浆的量，准备在步骤2.6用于蛋白质沉淀离心机的容量不超过50％。 </li><li>如果收购了脑组织的质量是有问题的，评估的PrP C水平通过SDS-PAGE 16和免疫17基板事先准备。 </li></ol></li><li>分割脑匀浆成在分开的标记的塑料锥形管2等体积。加入盐酸胍（的3M溶液中1 PBS）中在任一pH值3.5或7.4至1的每个管:1的体积比向脑匀浆。 </li><li>检查两种溶液的pH值。用浓HCl将pH调节3.5衬底至pH的pH±0.05。确保其他基片的pH值保持在pH7.4±0.05，并根据需要在必要时用HCl或NaOH调节pH。避免通过明智加入酸或碱的过冲的pH值。 </li><li> 5小时旋转，在室温（RT）下在最终过端混合器解决方案。 </li><li>通过加入4体积的甲醇中以在每个锥形管的底物溶液中沉淀的蛋白质，涡旋拌匀，孵育在-20℃进行16-18小时。 </li><li>沉积物样品离心以13,000×g离心30分钟，在4℃。小心倒出并丢弃上清液，并且允许甲醇从粒料蒸发。使用棉尖涂药器，以协助吸收甲醇紧贴于管的内部。不要让小球过度博士y和一旦甲醇不再可见，进行到下一个步骤。 </li><li>悬浮颗粒的蛋白质在转换缓冲。使用的转换缓冲等于脑匀浆的起始原料分配到各管中，在步骤2.2的量的量。 注:该转换缓冲用于从两个pH为3.5和7.4，经处理的衬底准备重悬蛋白质粒料在上面的步骤1.2（其中包含洗涤剂的低含量和生理pH）中定义的溶液，这一点至关重要。 </li><li>在cuphorn超声仪简要超声基板解决方案（10秒，约30％的最大功率），分装成0.5 - 1.0毫升卷标记好的1.5 ml离心管。在每一基底的等分试样进行质量控制的免疫印迹（步骤2.9）。存储其他等分在-80°C，直到准备进行CER检测（第4）。 </li><li>执行关于在使用前CER基板对（ 图2）的质量控制。这些措施确保了CER基板将providË最佳效果转化研究。 <ol><li>确保在两个基板可比数额的PrP 的C。在10-25微升每个衬底通过SDS-PAGE 16和免疫印迹17比较朊病毒水平。如果蛋白水平在pH为7.4和3.5的基材是彼此的10％之内〜，衬底对适合在CER研究用途。 注:朊蛋白免疫印迹不应该表现出广泛的PrP的Ç降解的证据。对朊蛋白的免疫反应应该是主要&gt; 20 kDa的。频带小于这个分子量可以降解产物。带型应该是基板对之间大致相同。 </li><li>作为朊下在两个基板应的PK敏感，治疗10-25微升每个衬底具有的PK在100微克/毫升的最终浓度和消化的样品以1000rpm在37℃下1小时的热振动筛。评估任何剩余的PrP信号通过SDS-PAGE 16和17的免疫印迹。底材具有朊病毒水库不适合在CER研究中使用。 </li></ol></li></ol> 3.准备TSE代理种子<ol><li>获得来自感兴趣物种TSE感染的脑组织及（重量/体积）匀浆中的1×PBS pH 7.4的使用在上面的步骤2.1中列出的方法制备的10％。 注:种子还可以从中枢神经系统以外的其他TSE感染的组织中产生，但是，通过从TSE感染的末梢组织衍生的种子转换脑源性底物的效率尚未被评估实验中公开的文献。 </li><li>等分所得匀浆（S）为100 - 在-80℃，直到准备300微升体积在0.5ml微量离心管并储存以执行CER测定。 </li></ol> 4. CER分析注:CER测定涉及从一种物种加入相对少量的PrP TSE的（设置在"种子"）成相对过剩的PrP C与另一个物种已经部分变性在任一pH值3.5或7.4（设置在未感染脑"衬底"）。以下扩展晃动潜伏期，朊蛋白的C模板驱动的转换由朊病毒TSE每个反应是由朊病毒水库剩余PK消化和免疫检测后的光密度信号进行评估。在先前在pH 3.5 与 7.4变性基板相比较的PrP 水库水平提供了物种屏障的量度。该测定过程的一个实验性的概述在图1中提供。 <ol><li>慢慢解冻未感染的CER基底对通过将它们放置在床上的冰（约1小时解冻时间）的顶部（测定需要预先在两个pH值3.5和7.4变性基板等体积）和TSE感染的种子的解决方案。 </li><li>一旦完全解冻，吸，然后驱逐通过每个底物液27G的注射器针头几次重新匀化它。简要超声仪处理10秒，约30％的最大功率每个TSE感染种子的解决方案。保留所有的解决方案在冰同时准备转化反应。 </li><li>标签5个别低结合，薄壁PCR每TSE试剂管被测试。 </li><li>通过加入5微升10％TSE感染种子溶液以95微升（一）CER基板的在pH7.4和（b）的CER基板在pH 3.5制备制备制备在这些管中转化反应。 <ol><li>对于每一个实验样品，准备在此前双方在pH为3.5和7.4变性CER基板对平行反应。 </li><li>通过实证确定优化TSE剂种子到未受感染的大脑基​​底的比率。常见种子:采用基片比率保持100微升的总反应体积，并包括1:99微升，2:98微升和5:95微升。对于大多数应用，该5:95微升种子:基板的体积比是合适的，并且所显示的内容在第是的协议。 </li></ol></li><li>准备每TSE剂3的对照样品被测试如下:（1）加入5微升10％TSE感染种子溶液以95微升的转换缓冲器作为输入朊病毒水库的控制;添加5μl的转换缓冲到在（B）的pH为3.5和（c）pH为7.4，作为非特异性的，非模板化的转换控制制备95微升底物。 </li><li>短暂涡旋振荡每一样品在其封闭PCR管以混合种子和衬底良好。用瞬时脉冲在低速台式微型离心机遵循以除去反应混合物截留在PCR管盖的任何体积。 </li><li>负载样品中单独标记的PCR管放入台式热振动筛配接受PCR管。摇动样品以1000rpm在37℃下24小时。 </li><li>以下晃动，添加十二烷基肌氨酸钠，以2％的终浓度（重量/体积）和PK为100微克/毫升的最终浓度。消化的样品以1000rpm在37℃下进行1小时的热振动筛。 ñOTE:增加肌氨酸到的朊蛋白的C PK消化样品转换后的辅助工具。非种子选手的样品（步骤4.5）的假阳性信号加入肌氨酸的几乎消除。 </li><li>加入SDS-PAGE样品缓冲液，热样品至95℃5分钟，并解决剩余的PrP 水库通过SDS-PAGE 16随后的免疫印迹17每个样品中。 注:在加入样品缓冲液和加热，样品可以被立即处理或样品可以储存在-20℃或-80℃之前，免疫印迹。使用NOVEX的NuPAGE凝胶和传输系统中生成这里介绍的所有数据。从CER测定各个样品分别用样品缓冲液，并根据制造商的说明还原剂。样品随后被加热到95℃5分钟和30微升各样品装入一个12％的Bis-Tris预制凝胶。 </li><li>计算作为度量的转换效率比（CER）的物种屏障。朊病毒水库的通过光密度17测量水平和由该pH为3.5的样品的划分pH 7.4的样品的总密度。乘以100，以产生一个百分比。 </li><li>从独立的实验重复编译数据，以产生一个平均的CER±标准为一种具有给定TSE剂偏差。 </li></ol>

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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=Lesion+Profiling+and+Subcellular+Prion+Localization+of+Cervid+Chronic+Wasting+Disease+in+Domestic+Cats.">Lesion Profiling and Subcellular Prion Localization of Cervid Chronic Wasting Disease in Domestic Cats.</a> Vet Pathol. , (2014).</li><li>Stewart, P., 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=Genetic+predictions+of+prion+disease+susceptibility+in+carnivore+species+based+on+variability+of+the+prion+gene+coding+region.">Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.</a> PLoS One. 7 (12), e50623 (2012).</li><li>Russell, W. M., Burch, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. Metheun. , (1959).</li></ol>

The authors have nothing to disclose.

为了顺利完成本协议的，应注意所用的基板准备（步骤2.1.2）和种子底物比对转化反应（步骤4.4.2）未受感染的脑组织付给的PrP C水平。根据我们的经验，大脑可以提取用作CER基板大幅期间验尸后，只要朊病毒C是本通过免疫印迹（ 图4）。事实上，一些自溶观察用于CER研究山猫的大脑。然而，来自这些大脑衍生基板孵育仍然导致朊病毒水库的形成。我们推测，?...

传染性海绵状脑病（传染性海绵状脑病，朊病毒病）是一组的致命的神经变性疾病与影响各种动物和人的延长潜伏期。的TSE的推定病原体包括主机朊病毒蛋白（PRP）的错误折叠的异构体，其能够自我繁殖由朊病毒（PrP的C）的正常细胞形式的模板驱动转换成感染性的，疾病相关的形式（PRP TSE）积聚在受感染的主机1的中枢神经系统组织。传染性朊病毒哺乳动物一般从主机到主机传输的物种特异性的方式，这已经引起了"TSE物种屏障"限间传播的事件2的概念。朊病毒物种屏障的生物因素没有得到很好地理解。传染性的PrP TSE和主机的PrP C C之间的氨基酸序列相似性一个强烈影响是否发生转换3-5，但仍不足以解释在体内 6,7观察到的所有朊病毒传播事件。 因此，TSE物种屏障特性在很大程度上依赖于动物攻击研究:曝光从给定的物种幼稚动物朊病毒从另一个并测量产生的温育时间，疾病发作和发作率的传输效率的指标。小鼠表达的PrP C来自异源物种也可用于这种类型的研究8。用转基因小鼠生产和长期朊病毒生物测定，以及使用动物的伦理方面的考虑相关的成本，是阻碍了TSE物种界限的实验研究。人类物种屏障传染性海绵状脑病的评估依赖于设计，以表达人类的PrP C小鼠。这些小鼠需要很长的潜伏期屈从于人类的传染性海绵状脑病或修改吨他的人朊蛋白C分子迅速发病9。对非人类的TSE在这些小鼠中的潜伏期可能超出正常小鼠寿命使得有挑战性阴性结果的解释。非人灵长类动物也已被用于作为代理为研究人类物种的障碍，但这些研究是充满其他类型的动物实验和非人类灵长类动物的相同的挑战可能无法精确地概括疾病，因为它进入在人类宿主。 动物生物测定仍是"金标准"方法测量物种一个TSE的易感性，但障碍，成本和这些活的动物研究的伦理学已迫使调查的替代品。许多在体外实验的基础上，评估主机的PrP C中的转换蛋白酶K（PK）耐药状态（PRP 水库 ）时，由朊病毒TSE的种子，已经开发并用于研究TSE SPECIES障碍10-12。 在体外测定法的实例包括无细胞转化测定法，蛋白质错误折叠环扩增（PMCA），以及转换效率比率（CER）测定10-14。而没有这些测定的考虑涉及自然感染后的物种障碍帐户外围因素的影响，均可能是有用的，以确定潜在易感宿主为传染性海绵状脑病。 这里，我们提出的协议的CER测定，其中从正常脑匀浆衍生2变性的PrP C底物在台式朊转化反应中使用（ 图1）13。朊病毒下在变性在pH7.4基板只能被转换为朊病毒水库由朊病毒龄接种到在没有一个物种屏障14进行反应。与此相反，朊病毒C在另一基底变性，在pH 3.5允许它被转换为朊病毒水库以下孵化与朊病毒TSE来自任何物种，并作为转换的控制。到朊病毒水库 pH 7.4的衬底相在该pH值3.5底物转化的PrP 的C的比率提供了物种屏障的量度。我们已发现，CER测定所预言的已知的实验室小鼠的物种障碍，各种传染性海绵状脑病，并已用于测定在努力预测众多哺乳动物种，包括大角羊，慢性消耗性疾病（CWD）和其他的TSE 14的物种障碍， 15。研究者感兴趣的一个工具，允许TSE物种界限或PrP的Ç-to-RES朊病毒转化评估快速筛选会发现这种方法非常有用。

<table><tbody><tr><td>12% Bis-Tris SDS-PAGE gels</td><td>Life Technologies</td><td>NP0342</td><td></td></tr><tr><td>0.5 mm Zirconium oxide beads</td><td>Next Advance</td><td>ZROB05</td><td>Other varieties of beads are also effective</td></tr><tr><td>Antibodies</td><td>various suppliers</td><td></td><td>Select appropriate primary and secondary antibodies for immunoblot detection of PrP&lt;sup&gt;res&lt;/sup&gt; from species of interest</td></tr><tr><td>Bead homogenizer</td><td>Next Advance</td><td>BBY24M</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>369434</td><td>High speed with temperature control</td></tr><tr><td>Conical tubes</td><td>any brand</td><td></td><td></td></tr><tr><td>Cotton-tipped applicator</td><td>Uline</td><td>S-18991</td><td></td></tr><tr><td>Cuphorn sonicator</td><td>Heat Systems-Ultrasonics</td><td>W-380</td><td>Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td></tr><tr><td>Densitometry software program</td><td>UVP</td><td>Vision Works LS Image Acquisition and Analysis software</td><td>Other programs, such as NIH ImageJ, will also work</td></tr><tr><td>Dounce homogenizer</td><td>Kimble Chase</td><td>885300</td><td></td></tr><tr><td>End-over-end mixer</td><td>Labnet International</td><td>H5600</td><td></td></tr><tr><td>Ethylenediaminetetra acetic acid</td><td>Boston Bioproducts</td><td>P-770</td><td>Hazardous chemical: eye irritation</td></tr><tr><td>Guanidine hydrochloride, 8 M</td><td>Thermo Fisher Scientific</td><td>24115</td><td>Hazardous chemical: acute toxicity, skin irritation, eye irritation</td></tr><tr><td>Heating block</td><td>Fisher Scientific</td><td>11-718</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>435570</td><td>Hazardous chemical: strong acid</td></tr><tr><td>Lithium dodecyl sulfate sample buffer, 4x</td><td>Life Technologies</td><td>NP0008</td><td>Hazardous chemical: skin &amp;amp; respiratory irritation, serious eye damage, flammable solid</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A454-4</td><td>Hazardous chemical: acute toxicity, flammable liquid</td></tr><tr><td>Microcentrifuge tubes</td><td>any brand</td><td></td><td></td></tr><tr><td>Mini-centrifuge</td><td>Labnet International</td><td>C1301</td><td></td></tr><tr><td>N-lauroyl-sarcosine (sarkosyl)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L-5125</td><td>Hazardous chemical: acute toxicity, skin irritation, eye damage</td></tr><tr><td>Nonidet P-40</td><td>Amresco</td><td>M158</td><td>Hazardous chemical: skin irritation, eye damage</td></tr><tr><td>SDS-PAGE gel system</td><td>Life Technologies</td><td>NuPAGE electrophoresis system</td><td>Other SDS-PAGE systems will also work</td></tr><tr><td>PCR tubes (low-binding)</td><td>Axygen</td><td>PCR-02-L-C</td><td></td></tr><tr><td>Pestle homogenizer</td><td>Fisher Scientific</td><td>03-392-106</td><td></td></tr><tr><td>pH meter</td><td>Sentron</td><td>SI600</td><td></td></tr><tr><td>Polyvinyldifluoride membrane</td><td>Millipore</td><td>IPVH00010</td><td></td></tr><tr><td>Proteinase K</td><td>Promega</td><td>V3021</td><td>Hazardous chemical: skin &amp;amp; eye irritation, respiratory sensitisation, organ toxicity</td></tr><tr><td>Reducing agent for SDS-PAGE samples, 10x</td><td>Life Technologies</td><td>NP0009</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D6750</td><td>Hazardous chemical: acute toxicity</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Thermo Fisher Scientific</td><td>28364</td><td>Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td>Hazardous chemical; strong base</td></tr><tr><td>Syringe</td><td>BD Biosciences</td><td>various</td><td>Use syringe size appropriate to volumes of substrate to be homogenized</td></tr><tr><td>Syringe needles</td><td>BD Biosciences</td><td>various</td><td></td></tr><tr><td>Thermoshaker (PCR tube shaker)</td><td>Hangzhou All Sheng Instruments</td><td>MS-100</td><td></td></tr><tr><td>Tris base</td><td>Bio Basic</td><td>77-86-1</td><td>Hazardous chemical: skin, eye, respiratory irritation</td></tr><tr><td>Triton X-100</td><td>Integra Chemical Company</td><td>T756.30.30</td><td>Hazardous chemical: acute toxicity, eye irritation</td></tr><tr><td>Vortexer</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr></tbody></table>

assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

成功使用CER测定的在很大程度上取决于在转化反应中使用的底物对的质量。出于这个原因，下面的过程以制备CER基板对，一个质量控制免疫应执行（ 图2）。对于两个基板，测定10-25微升通过免疫印迹。的PrP C应该容易检测在每个衬底和朊病毒C水平必须接近两者是相同的。免疫反应性将主要​​高于20 kDa的可见光，但较小频带也可以是显而易见的。根据我们的经验，更...

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

assessing-transmissible-spongiform-encephalopathy-species-barriers

Research

JoVE Journal

Medicine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

7.7K 次观看.  USGS National Wildlife Health Center. Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

视频: Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

科研

免疫与感染

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

在正常人脑分离的可溶性和不溶性朊蛋白低聚物

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

医学

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

朊病毒蛋白质错误折叠循环扩增

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

临床检查协议检测非典型和经典刮在绵羊

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

实时颤振诱发的粪便中 CWD 朊的转化检测

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

朊病毒安全实验室滑动测试

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

免疫组化检测异常朊蛋白

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform ...

神经科学

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

基于蛋白质的继承在酿酒酵母中的高通量筛选

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

生物学

Detection of Misfolded Prion Protein Aggregates in Mouse Brain Tissue Using Western Blotting

Take tissue homogenate from a mammalian brain that contains both normal prion proteins and misfolded prion aggregates responsible for neurodegenerative diseases.
Incubate with a proteolytic enzyme to digest the normal prions, leaving the resistant aggregates intact.
Treat the aggregates with a denaturing buffer at a high temperature to linearize the proteins and impart a uniform negative charge.
Load the sample onto an electrophoresis gel and separate the proteins into bands, which confirms the presence of aggregates.
Using an electric field, transfer the separated proteins onto a membrane.
Incubate the membrane in a blocking solution to mask non-specific binding sites.
Incubate with peroxidase enzyme-tagged antibodies, which bind specifically to the prion aggregates.
Wash the membrane to remove unbound antibodies, minimizing non-specific signals.
Add a chemiluminescent substrate and an oxidizing agent, enabling the peroxidase to oxidize the substrate and produce light.
Measure the light intensity to quantify misfolded prions in the homogenate.

评估传染性海绵状脑病物种界限与

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