We thank the following people: Jane Chu, Cameron Tran, James Lillard, Mafuz Khan, Masebonang Albert, Ken Rogers, and Syed A. Ali. Kateena Addae-Konadu was supported by UNCF/Merck Graduate Research Fellowship, American Medical Association Foundation, CRECD Grant 8R25MD007589-10, and NIH NIGMS MBRS Grant R25 GM058268. This work was supported by NIMHD grants 8G12MD007602, and 8U54MD007588, NIAID grant 1R21AI095150-01A1, Georgia Research Alliance grant GRA.VAC08.W, and Emory CFAR grant P30 A1050409.

外来体的分离和纯化过程的总体框图如图1中提供 。全血从健康志愿者的捐助者和未接受抗逆转录病毒theraoy参加埃默里大学的希望诊所和格雷迪健康系统的传染病计划HIV阳性者获得在佐治亚州亚特兰大。该研究获得埃默里大学医学院和莫尔豪斯学院的机构审查委员会。所有参与研究的人签署书面知情同意。 从血浆1.准备外来体的<ol><li> 人体血液采集与处理 <ol><li>收集10毫升外周血通过静脉穿刺在EDTA血液收集管（含有18毫克的钾EDTA）中，并轻轻颠倒5次混合。 </li><li>离心血液采集管在1000×g离心20分钟，在RT以沉淀血细胞。使用无菌吸管德的血浆部分（4-5毫升）转移到25毫升锥形管中。稀释血浆用10ml 1×PBS中。在对生物危险废物的适当标记的容器中丢弃血细胞（红细胞，白细胞，也被称为PBMC）。 注意:在未感染的供体的血液样品的情况下，PBMC可保留用于进一步使用（见步骤IV.2，下文）。 </li><li>储存血浆样品在4℃下短期（2-3天），或在-80℃下长期储存。 注:将冰冻的血浆样本，以4 摄氏度之前进一步处理。 </li></ol></li><li> 从血浆中的外来体分数的制备 <ol><li>离心机等离子体以10,000×g离心30分钟，在4℃以除去细胞碎片。使用无菌血清吸管给清零血浆上清液转移到一个干净25毫升超速离心管。丢弃在生物危害容器中的沉淀物。 </li><li>离心清等离子10万XG 2小时，在4℃，以除去大囊泡。小心地取出10万XG上清液吹打，并丢弃在生物危险废物。重悬在1ml的1X PBS的100000×g离心沉淀在一个干净的管中并在室温下孵育30分钟，轻轻地涡旋以移去和单独的颗粒。 </li><li>加入25毫升的PBS洗暂停100000 XG外来体/病毒颗粒。轻轻颠倒试管5次混合，然后再次离心以100,000×g离心2小时，在4℃。丢弃在生物危险废物容器中的PBS液冲洗。 </li><li>重悬100000×g离心沉淀在1ml 1×PBS中并在室温下孵育30分钟轻轻涡旋撞出和溶解沉淀。 注意:如果这是不可能直接进行到碘克沙醇梯度步骤，存储所述粒料，含有外来体和病毒颗粒，在4℃下1-2天，直到碘克沙醇梯度步骤。 </li></ol></li></ol> 2.净化外来体的<ol><li>生成6％-18％veloc碘克沙醇的使用双室梯度前装置，两者均梯度。 <ol><li>制备碘克沙醇试剂的6％和18％的溶液，供给作为在水中的60％的溶液中，通过稀释于PBS中。吸取5.5中毫升18％溶液进入搅拌室的，和吸管5.5中毫升6％溶液进入储存室。开启搅拌器，打开活塞，并且允许每个梯度流入14 ml的超速离心管。 注:要么立即使用或存放准备梯度O / N在4℃下才能使用。 </li><li>仔细层1毫升外来体/病毒溶液到每11个ml的梯度的顶端。离心机梯度在250000×g离心2小时，在4℃下，用SW40Ti吊桶式转子。 </li><li>标签12（12）1.5 ml微量离心管。从渐变的顶部除去1.0毫升并转移到管＃1中。转移顺序的其余1毫升馏分管2-12。 注:因此，最上面的级分将是＃1，底部馏分，＃12。圣矿石在4℃的梯度级分。纯化的外来体，其应在馏分1-3在梯度的顶部，贮存于4℃时是稳定的3-4周。 </li></ol></li></ol> 3.外来体的表征<ol><li> 乙酰胆碱酯酶活性测定 <ol><li>准备100毫米基板原料混合乙酰胆碱碘28.9毫克1毫升1X PBS中。商店基板股票在-20°C至1个月。 </li><li>准备10毫米彩色指标股混合苯甲酸39.6mg和碳酸氢钠15毫克于10毫升的1X PBS中。存储颜色指示剂股票在4℃下长达两个星期。 </li><li> 2:在100的比例制备测定试剂通过混合1×PBS，基板，以及彩色指示器5（例如，将10毫升的1X PBS + 200微升底物+500μl的颜色指示）。 </li><li>传送从各1.5 ml的梯度级分管（标记为1-12，从程序III.3）50微升到96孔microtite的孔- [R盘。准备两个孔的每个梯度样品。 </li><li>首先使2000μU/ ml的乙酰胆碱酯酶的股票在PBS准备一套标准。使11（11），2倍这个股票的连续稀释，并添加50微升每种稀释液至微量滴定板的单个孔，使得标准＃1 = 2000μU/毫升，＃2 = 1000μU/毫升，＃3 = 500μU/ ml的，等等，直到＃12 = 0.98μU/毫升。因此，12的AchE标准将占用一个96孔测定板的单个行。 </li><li>加入200μl测定试剂混合物的每个孔中，孵育20分钟（在黑暗中）在RT下，以使有色反应产物的发展。测量在450nm使用荧光酶标仪在波长AChE活性。 注意:在起动外来体纯化后的剩余材料的百分比是由普通等离子体胆碱酯酶法检测。 </li></ol></li><li> 免疫印迹分析 <ol><li>在梯度分数1-12分离蛋白（从的程序，URE 2.1.3）通过SDS-PAGE在4-20％的Tris-HCl预制凝胶在100V×1小时。在凝胶转移蛋白根据生产商的说明使用电印迹转移细胞硝酸纤维素膜上。允许转移到印迹12-16小时（O / N）为350V。 </li><li>从印迹装置取出膜和洗涤在Tris-缓冲盐水（TBS）中5分钟。用5％脱脂牛奶在TTBS（TBS含0.1％吐温20）1小时通过振荡在RT方框。 </li><li>孵育在5％脱脂牛奶特异于外来体（CD9，CD45，CD63）或HIV-1衣壳蛋白（p24的）第一抗体的膜，在4℃振荡12-16小时（O / N）。对于原发性免疫印迹稀释抗体的范围是从1:2000 -1:5000。 </li><li>用1洗印迹在TTBS 20分钟，然后孵育:2000稀释的辣根过氧化物酶（HRP）缀合的抗IgG抗体（二次抗体），在5％的脱脂牛奶1小时，在室温。洗净的印迹3次TTBS，每洗10分钟。 </li><lI&gt;孵育印迹用鲁米诺底物试剂根据制造商的指示，以产生化学发光信号从HRP标记蛋白检测使用具有CCD照相机的电子成像系统的化学发光信号。 </li><li>将图像保存为可以在Adobe Photoshop中查看TIFF文件。执行使用ImageJ软件（国立卫生研究院，马里兰州贝塞斯达）乐队的密度分析。 </li></ol></li><li> 细胞因子分析 <ol><li>在此之前的细胞因子测定，破坏免疫复合物，通常是存在于人血浆中的酸解离并裂解外来体通过去污剂处理。分析这两个启动血浆样品纯化外来体的准备。 <ol><li>至100μl人血浆添加100微升0.33当量盐酸，在37℃进行1小时。加入100微升0.33 1N氢氧化钠中和酸处理的血浆。添加的Triton X-100，以两者中和的血浆和纯化外来体的样品，给音响的1％的最终浓度，以引起外来体的裂解。 </li></ol></li><li>对于该测定法（荧光基于珠子的程序），使用磁珠预先涂覆有抗体由生产。使用抗人细胞因子抗体包被的珠的定制设计的面板; 在表1中列出。涡流从所述细胞因子测定试剂盒的抗体-包被的磁珠，30秒，并添加50微升珠96孔平板的每个孔在测定中使用。 </li><li>制备血浆标准缓冲液（均包含在细胞因​​子测定试剂盒）的稀释系列标准中的细胞因子测定试剂盒使用说明书中所述，以产生8点的标准曲线。 </li><li>添加150微升1X洗涤缓冲液（由试剂盒）到每个孔，并且洗涤用磁珠垫圈珠，如手册中所述。添加25微升血浆测定缓冲液（从盒）至各孔中。添加25微升标准品和样品，以在该测定中使用的所有孔中。加入25微升血浆样品缓冲液阴性对照孔中。密封，并在室温摇动板以700rpm进行60分钟和孵育O / N在4℃下。 </li><li>添加150微升1X洗涤缓冲液到每个孔，并且洗涤该板（如在步骤3.3.4，上文）。添加25μl的抗体检测到每个孔中，密封并在700rpm摇动板在室温30分钟。重复洗涤步骤。 </li><li>加入50微升链霉溶液（SAPE，从盒）到每个孔中，密封并在700rpm摇动板在室温30分钟。重复洗涤板在3.3.4。 </li><li>添加120微升读数缓冲液（从盒）到每个孔中，并摇动在实验室桌面摇床在700rpm进行5分钟，在室温以允许荧光信号来开发。读板放在读板器按制造商的说明。 </li><li>为了解释分离过程期间丢失外来体的量，可使用下列公式:[（原件读取/血浆体积）×66.6 =调节细胞因子读数。 </li></ol></li></ol> 4.检测方法的免疫调节潜力<ol><li> 培养基与外来体贫胎牛血清制备 <ol><li>离心机500毫升胎牛血清（FBS）中的10万xg离心20小时，在4℃以沉淀污染外来体和微泡。小心地取出并保存外来体耗尽FB​​S上清。丢弃在生物危险废物的颗粒。 </li><li>结合外来体贫FBS的上清液用500ml罗斯韦尔园区纪念研究所1640（RPMI 1640）培养基的20％。过滤通过0.45μm膜过滤器的介质。 </li><li>添加链霉素（100U /毫升），青霉素（100U /毫升），L-谷氨酰胺（2 mM计），HEPES缓冲的盐溶液（10μM），和IL-2（20U / ml）的经滤波的介质。 </li></ol></li><li> 细胞培养和外来体曝光 <ol><li>暴露外来体从健康诊所的志愿者得到的供体外周血单核细胞（PBMC）。在暂停PBMC制备培养基和计数使用细胞/颗粒计数器（根据制造商的标准方法）的细胞。 注:将PBMC上述等离子体制备过程中得到的（步骤1.2）。 </li><li>使用制备的介质从过程4.1.3，共培养3.0×10 6（PBMC）用1微克/毫升汇集外来体三（3）的HIV-1血清阳性或从三（3）的HIV-1血清反应阴性个体的总体积为1ml在12孔培养板（盖）的孔中。在37℃下孵育培养物48小时。 </li><li>准备与刀豆蛋白A处理未处理的外周血单个核细胞培养物和培养（ConA的; 5微克/毫升）以上（4.2.2），如作为阴性和阳性对照，分别。孵育所有PBMC培养48小时，在37℃。 </li><li>之前立即收集细胞，准备以下荧光标记的单克隆抗体的稀释度:的Alexa Fluor 700标记的抗CD3（1:400稀释），allophycocyANIN（APC）/花青7（Cy7的）标记的抗CD4（1:400），多甲藻素叶绿素蛋白复合物标记的抗CD4（1:400），V450标记的抗CD8（1:400），生物素标记的抗CD45RA（1:1000），藻红蛋白（PE）/ Cy7的标记的抗CD62L（1:1000），PE /花青5（Cy5的）标记的抗CD38（1:200），PE-德克萨斯红 - 标记的抗生蛋白链菌素（1:2000），和PE / Cy5标记小鼠IgG1K同种型对照（1:200）。 </li><li>在48小时的潜伏期后，收获PBMC。洗涤的PBMC在PBS中在4℃下温育除去外来体和细胞染色1小时与个别荧光染料标记的抗体。通过流式细胞仪分析染色的PBMC量化趋化因子的表达（如21）。 </li></ol></li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Nef+Protein+Targets+CD4++T+Cells+for+Apoptosis+by+Interacting+with+CXCR4+Surface+Receptors.">Extracellular Nef Protein Targets CD4+ T Cells for Apoptosis by Interacting with CXCR4 Surface Receptors.</a> Journal of Virology. 78, 3099-3109 (2004).</li><li>Huang, M. B., Jin, L. L., James, C. O., Khan, M., Powell, M. D., Bond, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Nef-CXCR4+Interactions+Important+for+Apoptosis+Induction.">Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction.</a> Journal of Virology. 78, 11084-11096 (2004).</li><li>Konadu, K. 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The authors declare that they have no competing financial interests.

慢性免疫激活（CIA）和CD4 + T细胞耗竭是HIV-1感染的两个重要特征。他们已经被确立为预测的发病机制，与美国中央情报局是最好的预测。然而，驾驶慢性全身免疫激活和CD4 + T细胞衰退的作用机制还没有完全阐明。我们和其他的实验室已经开发出坚定的证据表明，外来体从HIV-1感染细胞分泌起到两个标志的作用。 在两种组合物和细胞外囊泡的功能继续关心导致了各种方法外来?...

在HIV-1流行继续在世界各地都有一个显著的影响。截至2013年，全球约有3500万人感染艾滋病毒，而这些210万新感染的个体1。预防战略和增加获得抗逆转录病毒疗法已在减少整体收购艾滋病毒有帮助。然而，个别人群仍面临上升收购HIV 1。因此，有必要继续努力来解决这一流行病。 其中一个HIV疾病进展的最强预测的是慢性免疫激活（CIA）2-10。持续高检测水平的细胞因子和T淋巴细胞的表面上表达升高的标记限定，CIA已被归因于:i）连续的树突状细胞产生I型IFN 11的类型; （二）直接免疫激活HIV蛋白达，NEF和gp120的12驱动; （三）易位相关的肠道免疫细胞6细菌蛋白质。然而，确切的机制（S）潜在慢性，在HIV感染的全身免疫激活仍有待完全阐明。 我们的研究小组和其他人证明外来体的艾滋病发病15-18的作用。我们小组已确定的Nef蛋白从感染的细胞中分泌的外来体15，和外来体的Nef（exNef）存在的HIV感染的个体中的纳克水平18在血浆中。我们已经表明，旁观者的CD4 + T细胞暴露于exNef导致活化诱导的细胞死亡取决于CXCR4途径19，20，另外，单核细胞/巨噬细胞难治exNef诱导的细胞凋亡，但表现出改变的细胞功能和细胞因子的表达。最近，我们集团已经显示出外来体从HIV感染者的血浆中分离出含有多种促炎细胞因子。福rther，诱导CD38表达对天真和中央记忆CD4 +和CD8 + T细胞的HIV感染患者暴露于血浆来源的外来天真的外周血单核细胞。这可能有助于全身性炎症，并通过旁观者细胞活化21病毒的繁殖，并表明外来体发挥在HIV发病机理中显著作用。 在调查外来体在HIV发病机理中的作用，一个挑战是发展技术以来自HIV颗粒，同时保持外体含量有效地分离外泌体，以及它们的功能免疫调节能力。几种方法已经被描述用于从细胞培养物和生物流体22,23分离外泌体。因为它们的小尺寸和低的密度（外来体漂浮在1.15的密度- 1.19克/毫升），差分超速离心和/或超滤是最常用的技术为外来体隔离23。但是，艾滋病毒感染细胞培养上清和病人的血浆同时包含外来体和HIV-1病毒颗粒。外来体和HIV-1的颗粒在尺寸和密度非常相似。另外，以独特的外体标记，如CD63，CD45，CD81和表达的优势，外来体一直在使用免疫亲和捕获方法23隔离。这个过程可以从外来体分离病毒。然而，这种技术的缺点是紧密附接抗体与纯化的外来体，这可能干扰在培养外来体的免疫调节的潜能的评估。 为了解决这些局限性，我们开发了用于使用碘克沙醇的速度梯度来自HIV颗粒在人血浆中由Cantin改性和同事22外来体的纯化过程。外来体被发现在低浓度偏析碘克沙醇梯度/上层级分，而病毒颗粒分离在高密度/较低的分数。病毒颗粒通过P24 ELISA检测和使用外来体标记的乙酰胆碱酯酶，CD9，CD63和CD45被确定外来体。收集上层低密度部分包含这是不含有HIV-1 P24污染外来体。艾滋病毒颗粒在人体血浆中外来体的高效纯化和分离方法可以从人血浆以及他们的免疫调节潜力的调查和外来体在HIV-1发病机制中的诊断和预后价值来源的外来内容的准确检查。

<table border="0" cellpadding="0" cellspacing="0" width="506">
	<tbody>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">BD Vacutainer EDTA tubes (10ml)&#160;</td>
			<td style="width:92px;">Becton Dickinson</td>
			<td style="width:101px;">368589</td>
			<td style="width:127px;">pink top tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Lymphoprep Ficoll reagent</td>
			<td style="width:92px;">Cosmo Bio</td>
			<td style="width:101px;">AXS-1114545</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Optiprep iodixanol reagent</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">D1556</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">14ml ultracentrifuge tubes</td>
			<td style="width:92px;">Beckman Coulter</td>
			<td style="width:101px;">344060</td>
			<td style="width:127px;">ultraclear tubes</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Gradient Former Model 485</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">165-4120</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Acetylthiocholine iodide</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">1480</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Benzoic Acid</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">D8130</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Sodium Bicarbonate</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">S5761</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Acetyl Cholinesterase</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">C3389</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">96-well clear microtiter plate</td>
			<td style="width:92px;">Medical Supply Partners</td>
			<td style="width:101px;">TR5003</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">SpectraMax 190 microplate reader</td>
			<td style="width:92px;">Molecular Devices</td>
			<td style="width:101px;">190</td>
			<td style="width:127px;">Fluorescent plate reader</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Criterion Gel Electrophoresis Cell</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">165-6001</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Transblot Gel&#160;</td>
			<td rowspan="2" style="width:92px;">BIO-RAD</td>
			<td rowspan="2" style="width:101px;">170-3910</td>
			<td rowspan="2" style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Transfer Cell</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Tris-HCl Criterion precast gels</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">567-1093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Anti-CD45 antibody&#160;</td>
			<td style="width:92px;">Abcam&#160;</td>
			<td style="width:101px;">Ab10558</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">CD63 Antibody (H-193)</td>
			<td style="width:92px;">Santa Cruz Biotech, Inc.</td>
			<td style="width:101px;">SC-15363</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">CD9 Antibody (H-110)</td>
			<td style="width:92px;">Santa Cruz Biotech, Inc.</td>
			<td style="width:101px;">SC-9148</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Rabbit pAb p24 HIV-1&#160;</td>
			<td style="width:92px;">ImmunoDX, LLC</td>
			<td style="width:101px;">1303</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Nitrocellulose membrane&#160;</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">G1472430</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Tris Buffered Saline</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">170-6435</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">HRP-conjugated IgG (H+L) secondary antibody</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:101px;">31460</td>
			<td style="width:127px;">Goat-Anti-Rabbit</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">HRP-conjugated IgG (H+L) secondary antibody</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:101px;">31430</td>
			<td style="width:127px;">Goat-Anti-Mouse</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Western Blotting Luminol Reagent</td>
			<td style="width:92px;">Santa Cruz Biotech, Inc.</td>
			<td style="width:101px;">SC-2048</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">GE LAS-4010 Imager</td>
			<td style="width:92px;">GE Healthcare</td>
			<td style="width:101px;">LAS-4010</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">Human Procarta Cytokine Immunoassay Kit</td>
			<td style="width:92px;">Affymetrix&#160;</td>
			<td style="width:101px;">N/A</td>
			<td style="width:127px;">Custom immunoassay panel</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Bio-Plex 200 Immunobead Reader</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">171-000201</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Coulter Z2 Particle Counter</td>
			<td style="width:92px;">Beckman Coulter</td>
			<td style="width:101px;">383552</td>
			<td style="width:127px;">Cell counter</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">Alexa Fluor 700-labeled anti-CD3</td>
			<td style="width:92px;">BD Bioscience (UCHT1)</td>
			<td style="width:101px;">300424</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">APC/Cy7-labeled anti-CD4</td>
			<td style="width:92px;">Biolegend (OKT4)</td>
			<td style="width:101px;">317418</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">PerCP-labeled anti-CD4</td>
			<td style="width:92px;">BD Bioscience (RPA-T8)</td>
			<td style="width:101px;">550631</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">V450-labeled anti-CD8</td>
			<td style="width:92px;">BD Bioscience (RPA-T8)</td>
			<td style="width:101px;">560347</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">Biotin-labeled anti-CD45RA</td>
			<td style="width:92px;">BD Bioscience (HI100)</td>
			<td style="width:101px;">555487</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">PE/Cy7-labeled anti-CD62L</td>
			<td style="width:92px;">Biolegend (DREG-56)</td>
			<td style="width:101px;">304822</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">PE/Cy5-labeled anti-CD38</td>
			<td style="width:92px;">Biolegend (HIT2)</td>
			<td style="width:101px;">303508</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">APC/Cy7-labeled anti-HLADR</td>
			<td style="width:92px;">Biolegend (L243)</td>
			<td style="width:101px;">307618</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">PE-Texas Red-labeled anti-streptavidin</td>
			<td style="width:92px;">BD Bioscience</td>
			<td style="width:101px;">551487</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">PE/Cy5-labeled mouse IgG1K</td>
			<td style="width:92px;">Biolegend (MOPC-21)</td>
			<td style="width:101px;">400116</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">APC/Cy7-labeled mouse IgG2aK</td>
			<td style="width:92px;">Biolegend (MOPC-173)</td>
			<td style="width:101px;">400229</td>
			<td style="width:127px;"></td>
		</tr>
	</tbody>
</table>

isolation of exosomes from the plasma of hiv-1 positive individuals

外来体是小囊泡从从它们的多种细胞类型的释放都组成型和刺激后30纳米至100纳米不等的大小。他们发现，在一些生物体液的和已知携带多​​种蛋白质，脂质和核酸分子。原本以为要多一点水库细胞碎片，外来体调节生物过程和疾病中的作用也越来越赞赏。 几种方法已经被描述用于分离从细胞培养基和生物流体的外来体。由于它们的小尺寸和低的密度，差分超速离心和/或超滤是最常用的技术为外来体隔离。然而，HIV-1感染的个体的血浆中含有两个外来体和HIV病毒颗粒，其在尺寸和密度相似。因此，从人血浆中的HIV病毒颗粒的外来体的有效的分离已一个挑战。 为了解决此限制，我们开发了从Cantin 等改性的过程。 人，2008为来自HIV颗粒在人血浆中的外来体的纯化。碘克沙醇的速度梯度被用于外来体来自HIV-1颗粒的HIV-1阳性个体的血浆中分离。病毒颗粒确定P24 ELISA。外来体被确定的外来体标记物的乙酰胆碱酯酶的基础（乙酰胆碱酯酶）和CD9，CD63和CD45抗原上。我们的渐变过程产生游离病毒颗粒的外来体的准备。从人血浆外来体的高效净化使我们能够检查血浆衍生的外来体的含量，并调查它们的免疫调节潜力和其他生物学功能。

外泌体有效地从HIV-1阳性的人血浆纯化。分离的外来体，确定乙酰胆碱酯酶的活性，分隔在较低密度组分1-3在碘克沙醇梯度的顶部，而病毒颗粒，确定HIV-1抗原的p24 ，隔离在高密度级分（10-12，底部附近）。外来体的存在下通过的外来体标记物的AChE，CD9，CD45，CD63和，并用电子显微镜（图2）的免疫印迹鉴定进一步证实。 <p class="jove_content" fo:keep...

Watch this Scientific Journal Video about Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals at JoVE.com

从 HIV-1 阳性个体血浆中分离外泌体 |免疫学和感染|视频

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

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17.2K 次观看.  Morehouse School of Medicine. Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

视频: Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant &beta;-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2 used the ab initio algorithm 3 accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.

免疫相关动态三级的HIV - 1 V3环冠R2的序列结构特点的评估从头折叠

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

科研

免疫与感染

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

HIV - 1使用转速依赖的慢病毒表达载体的绿色荧光蛋白阳性细胞的特定标记

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

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 ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

HIV-1 Gag的可视化绑定到巨人的单片囊泡（GUV）膜

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...