从威康信托基金会通过资助，这项工作成为可能。 ，JM（080964）和SCW（080948）支持由威康信托基金会奖学金和DT由马拉维利物浦威康信托基金会助学金。

1。样品采集血小板可以很容易地通过激活温度，搅拌或存储，因此，他们应该小心处理。使用真空采血管收集血液血小板制剂在大多数临床情况是不可避免的，但应慎重考虑吸可能会导致血小板活化。由于我们的研究医院的临床使用的协议，我们的样本都是经过精心收集柠檬酸钠真空采血管。我们没有遇到任何问题，在我们以前的研究12血小板活化过早。我们收集血液血型O +个人血小板制剂，以尽量减少非特异性血型抗原聚集。捐助者也没有采取一些药物是众所周知的影响血小板功能的前7-10天用药。 1.1制备富血小板血浆（PRP） <ol><li>收集约5毫升的从疟疾天真的主机或2.5毫升的血液从CM或严重的疟疾（SM）患者中的柠檬酸钠真空采血管（BD产品编号36276）的全血。 </li><li>离心的全血在250×g离心10分钟，在室温下。不要离心，在4℃下，因为低温可能会导致血小板聚集。 </li><li>小心转移上清在一个干净的15毫升管多云血小板由于温度变化和物理冲击，很容易被激活，因此，应谨慎处理。避免摇动或任何生涩的动作管。 </li><li>使用一个纽鲍尔haematocytometer的计算和调整血小板悬液&gt; 300×10 3血小板/μl的健康献血者和&lt;150×10 3 CM使用1X磷酸盐缓冲液（pH值7.2-7.4; PBS）和SM患者的血小板/微升。请一定要选择一个高的稀释倍数，以缓解血小板计数，并确保其准确性。 </li></ol>注:疟疾拍拍的ients有血小板数量减少相比健康个人16,17,18。因此，血小板浓度为CM和UM根据为每个疟疾诊断组患者在血小板计数平均调整。 P.疟原虫丛生的表型是常见的在CM菌株和会表现出极强的亲和力，因此，降低血小板的浓度不影响丛大小或频率，因为血小板浓度是标准化的所有CM案件。 1.2制备贫血小板血浆（PPP） <ol><li>若要取得PPP，离心10分钟以上获得1500 XG覆检委员会的部分。大多数的血小板沉淀在试管底部的。 </li><li> PRP和PPP都可以储存在4℃长达两个星期。在理想的情况下，使用新鲜血小板结块检测。后8-10天血小板可能处于无效状态，可能凝集。 </li></ol> 2。帕拉斯ITE文化<ol><li>颗粒PRBC三次洗净用5-10毫升的RPMI 1640中，通过离心分离，在370×g离心5分钟。 注:在本协议中所有的文化开始从约1毫升红细胞压积（PCV），并维持在5％的血细胞比容。文化可以开始PRBC任何PCV与未感染的红细胞和媒体并作相应调整，以达到所需的血细胞比容。 </li><li>将PRBC在培养烧瓶中，并与标准的疟疾用25mM HEPES，5％Albumax II或10％血清和40微克/毫升庆大霉素，实现了5％的血细胞比容补充的RPMI 1640培养基补充。 </li><li>渗透物与92.5％的氮，2.5％的氧气和5％二氧化碳的混合物中的培养瓶中密封前和孵化。另外，气密蜡烛罐Trager的Jensen的方法都可以使用。 </li><li>对于PRBC由病人直接获得，孵化24-36小时，在37℃培养箱中培养瓶中，5％的CO 2，以获取成熟的寄生虫。 注:MOST实验室和文化适应的线是很容易保持在标准条件下的文化。有下面描述的简单的技术，可用于同步不同的寄生虫阶段保留生长速率。 </li><li>准备薄血涂片（如下文所述），并在光学显微镜下检查寄生虫成熟。 </li></ol> 3。薄血片玻片制备<ol><li>将约10-15微升血液磨砂载玻片上搁放在平坦的表面的一端上。 </li><li>直至血被均匀地分布在第二滑动的边缘的边缘的第二滑动触摸滴血。 </li><li>按住第二张幻灯片，在45°角，很快，但轻轻地，第一张幻灯片上没有施加太多的压力，将血液跨越的第一张幻灯片，使薄膜均匀分布的血液。空气干燥。 </li><li>浸幻灯片甲醇10秒。空气干燥。 </li><li>在2姬姆萨％ST浸幻灯片艾因至少10分钟。广泛冲洗，直到水运行明确。空气干燥。 </li><li>检查幻灯片使用90-100X油乳剂镜头，光学显微镜上</li></ol> 4。无性阶段的纯化和同步的纯化的成熟的P。恶性 PRBC结块检测是必需的步骤。事实上，只有成熟的寄生虫能够结合血小板受体，因为它是在这个生命周期阶段的相关配体的表达和从感染的红细胞的表面突出。有几种方法以纯化并同步的P的无性阶段恶性疟原虫。:晚无性阶段可以丰富Percoll梯度19，磁性细胞分选20或使用明胶沉淀21协议的描述在这里。山梨糖醇同步选择的环台22，在这之后的环培养24-26小时，以获得成熟的阶段（与没有NEED执行明胶浮选）。 4.1 Plasmagel浮选 Plasmagel浮选使用明胶样的解决方案，选择较低的重量把手形红细胞留在溶液中，而密集的环形式和花环沉底。此技术提供了高达90％的纯度的成熟阶段感染的红细胞，并可以用于户外及实验室分离物。但是，正如前面提到的，plasmagel浮选删除玫瑰花PRBC以及不成熟的阶段，可能会导致在这些样品具有高的玫瑰花结次数减少结块的频率。然而，由于缺乏玫瑰花PRBC浮选plasmagel后不会影响结块的测定，，因为玫瑰花结的PRBC和未感染的红细胞而结块是pRBCs结合介导的血小板的相互作用。配体参与了这两个特点是不同的，主要是补体受体对未感染的红细胞调解玫瑰花结的CR-1。<ol><li> prewarm琥珀酰明胶溶液和血清/ Albumax II培养基至37℃的水浴中。 </li><li>培养转移到一个干净无菌的15毫升管和沉淀细胞在室温下以370×g离心5分钟，通过离心分离。 </li><li>小心吸上清液，以便不打扰颗粒和悬浮细胞在同等体积的琥珀酰明胶和蛋白培养基。 </li><li>混合物转移到无菌的15毫升管，并允许放置在37℃培养箱中培养15-20分钟，或直到一个明确的划分可以看出，上下水位之间。 </li><li>小心转移上层新鲜无菌的15毫升管，加10毫升的新鲜RPMI。 </li><li>离心5分钟370 XG仔细弃上清。 </li><li>薄血涂片检查评估寄生虫和发育阶段，在光学显微镜下如第4节所述。 </li></ol>注:成熟PRBC范围的感染的红细胞dependi的60-90％之间纳克文化的初始寄生虫。对于成熟的寄生虫的比例较高，使用文化的初始寄生虫≥10％。 5。丛生含量<ol><li>使用一个纽鲍尔haematocytometer的计数并调整PRBC浮选plasmagel后得到的悬浮液1×10 8 PRBC个/μl。 </li><li>在一个新的1.5毫升管中，重新悬浮于5％的血细胞比容浓度为20μg/ ml的终浓度和PRP的总体积的10％，吖啶橙猪红细胞。 吖啶橙，而是可以被污染的寄生虫培养与使用前的5分钟25微克/毫升的溴化3,8 - 二氨基-5 - 乙基-6 - 苯基菲啶溴化。 例如，对于一个200微升反应混合物，加10微升成熟的PRBC 300×10 3 /微升PRP血小板从健康献血者或150×10 3从SM患者血小板/微升和170微升蛋白培养基，20微升。 注意:血小板:猪红细胞在最终反应的比率是20:1。这是很重要的，是允许的格言这个结块的样品的频率来确定。如果增加血小板减少对照，然后并非所有的聚集pRBCs可能有机会聚集。 </li><li>以同样的方式准备控制;未受感染的红血细胞和PRP，PPP PRBC确定血小板的作用在pRBCs结块。 </li><li>暂停转移到1.5-2.0毫升锥形螺丝帽小瓶。 </li><li>将小瓶在温和搅拌下，在10-12 rpm的转速下，在室温下轧制。 </li><li>检查丛形成由准备湿滑动的通过吸取10μlPRBC±血小板混合在玻片上，盖上玻璃条，并检查下荧光。 </li><li>可以做到在15-20分钟的时间间隔采样，共120分钟。一丛被视为合共三个或更多的感染红细胞。 </li></ol>

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Clin. Pathol. 123, 172-183 (2005).</li></ol>

没有利益冲突的声明。

我们已经描述了用于研究临床分离株中直接在字段中的PRBC血小板介导的结块检测。介导的血小板结块， 体育的特征行为恶性临床分离株，已被认定为一个独特的粘接型，频繁CD36结合菌株12,23-24。我们已经用这种检测，以确定三个要点:1） 在体外聚集型体育强恶性疟原虫分离出与疾病严重程度和诊断2）新的受体P-选择素GABA介导的血小板聚集一起CD36，3）CM患者中血小板减少的程度相关联的马拉维的儿童的，足以限制进一步形成PRBC团块体外 12。表现在第6条中所描述的，通过检查湿玻片制备血小板聚集形成的参与。高转速离心时，血小板可从PRP隔离为了尽量减少血型抗原反应，但是，我们使用整个PRP，以尽量减少过多的处理，并从高速离心血小板活化的过早。血小板活性可以测定血小板聚集试验，使用弱血小板激动剂，肾上腺素或二磷酸腺苷（ADP）25中所描述的26。 有几个方面的先前已被检测差的特点，其中之一是选择的PRP源和测定的结果，血液中的抗原组的效果的重要性。我们准备从个人谁是O型血+ PRP，并没有采取任何药物治疗，在过去的7-10天抽血之前，一些药品会影响血小板的行为。 其他因素可能会影响结块成功包括猪红细胞压积，寄生虫结块的检测水平和长度。使用高血细胞比容和血小板的共同UNT这将是很难说的集群是否是真正的一丛，或过多的脑细胞是否仅仅是坐在一起的，因为他们是密密麻麻的15。还结块通常随孵育时间较长。这些都是有用的指引，设计时，应考虑聚集研究。 我们发现，从不同的临床组的样本可以并行使用PRP从相同的供体，如果它是可以这样做的分析，但一般限于在有限的资源的设置，例如现场输血池。因此，很难有一个单一的O +捐助规范检测。因此，我们确定了几个O +血型相容性捐助方确保尽可能。大样本的大小的情况下，使用多个捐助者也是有帮助的，使献血负担上只有一个人不落。 15-20分钟的采样点是更加可行的，如果一个单一的PE者之前进行检测，让检测动力学湿幻灯片的准备和执行没有采样时间重叠的。最受欢迎的微观数据是视觉和有些客观的，因此，重要的是，验证了细胞计数超过一个人。在这种情况下，处理在一个时间的一个样本可以允许三到四个样品取决于个人效率的完整的分析。 可以使用临床和实验室菌株进行介导的血小板结块。实验室线，它是建议用来最大限度地提高CD36的结合寄生虫结块频率。该试剂盒可以耦合和其他凝集试验，如玫瑰花或用于聚集抑制实验，将启用的血小板上的受体，可能介导PRBC绑定，知之甚少及检验的这种表型方面的研究。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Invitrogen</td>
 <td>21870-092</td>
 <td></td>
 </tr>
 <tr>
 <td>Acridine orange</td>
 <td>Invitrogen</td>
 <td>A3568</td>
 <td>10 mg/ml</td>
 </tr>
 <tr>
 <td>Gelofusine</td>
 <td>B Braun</td>
 <td>Gelofusine</td>
 <td></td>
 </tr>
 <tr>
 <td>1 M HEPES</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td></td>
 </tr>
 <tr>
 <td>Gentamycin</td>
 <td>Invitrogen</td>
 <td>15750045</td>
 <td>50 mg/ml</td>
 </tr>
 <tr>
 <td>Albumax</td>
 <td>GIBCO</td>
 <td>1102-037</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium citrate vacutainer</td>
 <td>BD</td>
 <td>362760</td>
 <td>4 ml capacity</td>
 </tr>
 <tr>
 <td>Inverted Microscope</td>
 <td>Leica</td>
 <td>MD16000 B</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorescent microscope</td>
 <td>Leica</td>
 <td>EL6000</td>
 <td>Contains blue and green light fluorescent filters for acridine orange and ethidium bromide </td>
 </tr>
 </tbody>
</table>

a simple protocol for platelet-mediated clumping of plasmodium falciparum-infected erythrocytes in a resource poor setting

恶性疟原虫引起严重的疟疾感染的大多数。脑型疟疾（CM）的病理生理机制尚不完全清楚和几个假说被提出，包括机械性阻塞微血管由P.疟原虫寄生的红细胞（PRBC）。事实上，在其生命周期阶段的红细胞内，P.疟原虫具有独特的能力，修改受感染的红细胞表面由不同的粘接性能上红细胞膜表面抗原出口。这允许多个组织和器官的毛细血管后静脉微血管内皮细胞衬里的附着力PRBC封存。通过这样做，成熟形式的寄生虫避免变形的感染的红细胞的脾间隙2和限制他们的环境更有利的低氧分压。其结果是本SEQUEST配给，它是只未成熟无性寄生虫和配子体外周血中可以检测到的。 成熟的PRBC众多的宿主受体表达微血管床的细胞粘附和封存发生严重和不复杂的疾病。然而，一些证据表明，只有特定的胶粘剂表型很可能要与疟疾严重的病理结果。这种特定的宿主-寄生虫相互作用的一个例子已经证明在体外 ，细胞间黏附分子1 PRBC支持绑定特定的粘接性能的能力已被链接到开发脑型疟疾4,5。此外，胎盘已被确认，作为一个网站优惠PRBC积累在感染疟疾的孕妇，与chondrotin硫酸a以合体上线胎盘间隙的主要受体6。玫瑰花的PRBC tò未受感染的红细胞通过补体受体1（CD35）7,8也一直伴随着严重的疾病9。 其中最最近描述的第恶性疟原虫细胞粘附表型的能力PRBC 在体外形成血小板介导的团块。这样的PRBC团块的形成需要CD36，血小板的表面上表达的糖蛋白。另一个人的受体，gC1qR/HABP1/p32，不同的细胞类型，包括内皮细胞和血小板上表达，也被证明，以方便PRBC粘附血小板形成团块10。无论是聚集在体内发生尚不清楚，但它可能占马拉维儿童死于CM 11脑微血管血小板显着积累。此外，临床能力聚集在体外分离培养，直接关系到疾病的严重程度，马拉维12 </sup和莫桑比克患者13，（虽然不是在马连道14日 ）。 随着几​​个方面的的PRBC结块型差的特点，目前对这个问题的研究还没有跟上一个标准化的程序。这是一个重要的问题，因为已知的高的固有变异性在测定中15。在这里，我们提出了一个方法， 在体外介导的血小板结块P.恶性希望，它将提供一个平台，让其他群体一致的方法以调查这种表型在未来的研究中，提高认识的局限性。在马拉维为基础，我们提供了一种有限的资源设置专门的协议，具有的优点，新鲜收集的临床分离株的表型，可以进行检查，而不需要冷冻。

介导的血小板聚集试验，使用约70％的成熟阶段的第一个例子的浓缩后由plasmagel浮选恶性 如图2所示。一丛合共三个或更多的感染红细胞。染色，吖啶橙或溴化乙锭荧光显微镜可以通过可视化。吖啶橙荧光光谱相似，在502 nm处的最大激发和发射最大值在525 nm（绿色），而溴化乙锭在493 nm处的最大激发和发射最大值在620nm（若丹明染料相似，红色）。竹丛很容易发现，在显微镜下，正常光线下，他们出现细胞聚集，根据荧光斑点30分钟的时间点使用吖啶橙染色，吖啶橙或溴化乙锭染料，分别时， 如图2所示（绿色或红色）。较大的团块，较大的细胞集合体出现未正常光线和较大的斑点的绿色或红色下为各染料的荧光。由于染料的靶DNA和细胞核染色，血小板和未感染的红细胞，未检测到荧光显微镜下，由于其缺乏晶核。 PRBC的小团块4.7随机形成±1.6 pRBCs，/丛发生后30分钟。丛的大小显着增加一些团块中含有许多的PRBC /丛，不能被肉眼计数孵育120分钟后的平均&gt; 15 /猪红细胞团块。结块的表型的频率受感染的细胞的数量被计算为团块/ 1,000个感染的细胞中存在，在重复测定计数。 <img alt="图1" fo:content-width="3.5in" fo:src="/files/ftp_upload/4316/4316fig1highres.jpg" src="/files/ftp_upload/4316/4316fig1.jpg" /> 图1。工作流血小板聚集实验。ŗICH和可怜的血浆（A）和P.恶性疟原虫感染的红细胞文化（B）准备，然后结合结块测定（C）。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/4316/4316fig2highres.jpg" src="/files/ftp_upload/4316/4316fig2.jpg" /> 图2。由P.感染的红细胞聚集疟原虫实验室隔离开来。竹丛由HB3 0时，30分钟和120分钟，富血小板血浆（A）和可怜丛形成贫血小板血浆（B）。 含量限制有一些限制，具体影响血小板功能或影响结果，其中大部分都已经强调阿尔曼和Rowe 15。在这里，我们会提及一些潜在的问题，可能会遇到各色NT阶段的检测: <ol><li>临床分离株适应体外培养可能是一个挑战，因为有时需要较长的培养期，以达到足够高的寄生虫允许形成团块。 体育与的高抗原变异率粘接型疟 ，文化适应的寄生虫可能无法反映人口经过一段很长的时间在文化的原感染寄生虫。 </li><li>为了避免由于血型抗原的非特异性凝集，血小板捐助者应匹配的血型抗原的临床分离株中使用的相同的测定，或通用型血抗原O组使用。然而，个人的血型O型是罕见的马拉维等非洲遗址和血小板取自常用血型O +捐助者和血型必须匹配。 </li><li>在潮湿的玻片制备计数团块繁琐和具有挑战性的图像捕获的c英语学习者在不断运动。一般，猪红细胞堆积在盖玻片的边缘，或者他​​们往往会粘在一起，形成与真正的团块，可以很容易混淆的"假"团块。为了减少细胞的运动，使细胞沉淀物，在分析之前，在室温下15分钟。 </li><li>结块试验是对时间敏感的，因此，只允许每个人的时间以获得最佳的精度进行分析的一个样本。在两个不同的样品的采样时间点，可以很容易地重叠，导致不匹配的采样时间点之间的样品，如果在同一时间处理多个采样。在这种情况下，复杂的定量软件可以计算丛大小或交替细胞特异性抗体可用于分析组合物团块。虽然这些技术可以大大提高数据的意义，可能被用于该法是在资源贫乏，这样的技术和设备，是不容易获得。 </li></ol>

Watch this Scientific Journal Video about A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting at JoVE.com

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

此方法研究介导的血小板凝结的表型的<em&gt;恶性疟原虫</em&gt;感染红细胞（PRBC）临床分离株。这是由隔离和共同孵育富含血小板的血浆和猪红细胞的悬浮液。

一个简单的协议介导的血小板聚集<em&gt;恶性疟原虫</em&gt;受感染的红细胞在一个资源匮乏的设置

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

a-simple-protocol-for-platelet-mediated-clumping-plasmodium

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

18.7K 次观看.  Malawi-Liverpool-Wellcome Trust Clinical Research Programme. 此方法研究介导的血小板凝结的表型的恶性疟原虫感染红细胞（PRBC）临床分离株。这是由隔离和共同孵育富含血小板的血浆和猪红细胞的悬浮液。

视频: A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书"

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

分离<em&gt;恶性疟原虫</em&gt;晚期感染的红细胞由磁性装置

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

一个协议，用于分析丙型肝炎病毒复制

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

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