Name: an in vitro model for measuring immune responses to malaria in the context of hiv co-infection
Uploaded: 2015-10-06T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection at JoVE.com

C.A.M.F. and L.S. participated in protocol design, acquisition and analysis of data, and drafting of the article. 
The authors wish to thank Dr. Kain, Dr. Loutfy, Dr. Wasmuth, and Dr. Ayi for their contributions.
C.F. was supported by a CTN/Ontario HIV Treatment Network (OHTN) postdoctoral fellowship. L.S. is supported by an OHTN Junior Investigator Development Award. The present work was supported by a Canadian Institutes of Health Research (CIHR) operating grant (MOP-13721 and 115160), and a CIHR New Investigator Catalyst grant.

此协议需要捐助者的血清和红细胞用于寄生虫文化的招募和HIV（+）和未感染的捐助者外周血单个核细胞分离。机构审查委员会必须批准所有研究和所有捐助者必须提供之前抽血知情同意书。 警告:人类血液样本和人类疟原虫工作需要的预防措施。总是穿着白大褂，手套和工作在2级生物安全柜。在意外经皮接触到人类疟疾的情况下，报告给健康和安全的预防性治疗。额外的安全考虑也应到位与HIV（+）血工作。穿一回收盘白大褂。双人手套（顶级手套应胶乳）。执行所有操作在2级生物安全柜。不要使用任何玻璃或尖锐物体。不要使用抽吸。发生在virox溶液或漂白粉所有被污染的物品之前丢弃至少1小时的。洗virox和UV所有表面1小时以下使用。需要注意的是每家机构将需要遵循自己特定的生物安全法规。报告所有意外暴露于艾滋病病毒感染者的血液，以健康和安全评估和考虑可能的暴露后预防。 注:P的不同菌株恶性疟疾寄生虫存在。 ITG被用于这些实验，但不同的菌株可以使用。可在MR4网站13冻融恶性疟原虫的寄生虫优秀的说明。 1.制作RPMI-A为疟疾寄生虫文化<ol><li>使RPMI-0通过混合950 ml的DDH 2 O的的，的RPMI-1640粉末1包，6克的HEPES，2克碳酸氢钠，以及1.35毫克次黄嘌呤。 </li><li>解冻热灭活人血清来自两个不同的捐助者。 AB捐助者最好的，但任何可以使用，如果寄生虫生长在O型红细胞（RBC）。反转管混合。如果血清含有的颗粒物质或较厚自旋在2,000rpm，然后用0.45微米的过滤器单元进行过滤的液体部分。 </li><li>使用0.2微米的过滤器单元过滤器180毫升的RPMI-0，20毫升的人血清（从每个供体10毫升），0.5毫升10毫克/毫升庆大霉素。 注意:人血清会堵塞过滤器，以便一个以上的过滤器单元，可能需要。 </li><li>标记介质瓶用RPMI-A中，日期，和血清的来源。冷藏备用。冷藏时RPMI-A可能会变成浑浊。这是正常的，但云量增加是污染的迹象。 注:寄生虫的生长可能会在不同的供体血清而有所不同。这是在使用之前测试所有的人血清批次良好的寄生虫的生长是一个好主意。 </li></ol> 2.准备人红细胞寄生虫文化注:献血者应该是O型。 <ol><li>收集7-10毫升血液进入酸CIT率 - 葡萄糖（ACD）管中。写捐赠者的ID和收集日期的标签。 </li><li>直到需要血液储存在摄氏 4度。使用血液寄生虫培养1个月内。 </li><li>擦拭用70％乙醇的管的顶部。小心取出瓶塞并丢弃。传输血液入15ml管中。自旋为3分钟，在1000×g下。吸除去血浆。 </li><li>暂停红细胞与温暖的RPMI-0等体积。自旋5分钟，在1000×g下。吸取出白膜层，悬浮在5毫升RPMI-0，重复洗2次以上。 </li><li>除去上清液，并添加足够的RPMI-A以产生混合物，其是50％的RBC（体积）。 </li><li>储存在摄氏 4度，直到需要。 </li></ol> 3.维护寄生虫文化<ol><li>前期预热RPMI-A到37℃。 </li><li>放置解冻寄生虫（见MR4协议解冻过程）转换成T25烧瓶用5ml RPMI-A和75微升洗涤人RBC为〜3％血细胞比容。注意:血细胞比容测定红细胞的体积相比，总体积。计算血细胞比容测量填充红细胞到培养基加上红细胞的总体积的体积。假设解冻寄生虫将推动75微升红细胞。通过加入红细胞库存150微升（相当于加入75微升填充红细胞）向烧瓶共有150微升RBC入5毫升培养基，它等于3％150微升/ 5000血细胞比容（是微升×100（％）= 3％）。 </li><li>气的烧瓶中，持续30秒与寄生虫气体混合物（1％O 2，3％的CO 2，平衡N 2）。密封和向下放置烧瓶宽的部分，在37℃培养箱，以允许为最大的表面积为气体交换。 </li><li>要改变中，检查原虫（需要每天做的），小心地将瓶中，以不打扰RBC层。使用无菌拔出巴斯德吸管抽出培养基不影响血液层。 </li><li>要检查PArasitemia，从血液中去除层10微升样品，将其放置在载玻片上，并使用第二载玻片创建一个薄血膜。允许滑动晾干。 </li><li>放置4毫升新鲜的RPMI-A到烧瓶中，轻轻地混合，气体30秒，并将其放置在培养箱中在37℃。 </li><li>染色根据制造商的协议使用Hema3染色试剂盒的幻灯片。 <ol><li>为了达到最佳的染色，浸渍在滑动定影液10秒，溶液I，10秒，和溶液II为30秒。在水冲洗和离开载片干燥。 </li></ol></li><li>通过计数PfRBC的数目计算寄生虫血症（染色深紫色）对红细胞（染色粉红色）的总数。算共有300细胞以确保准确性。 </li><li>当寄生虫已经达到了5％的原虫，扩大文化到T75烧瓶，用40毫升RPMI-A和500微升红细胞。 </li></ol> 4.寄生虫同步注:t前一天他尝试通过与丙氨酸处理同步寄生虫文化。只有环阶段寄生虫和未受感染的红细胞将在本治疗。丙氨酸同步会给你一个纯粹的滋养体的文化，可以在共培养实验中使用的第二天。请务必先从含有大多数的环阶段寄生虫寄生文化。 <ol><li>通过混合8.01克丙氨酸（300毫摩尔）和0.365克三（10毫米）的入300ml双蒸2 O的制备丙氨酸溶液把pH值至7.4。过滤消毒使用0.2微米的过滤器单元。 </li><li>前期预热丙氨酸解至37℃。 </li><li>降速寄生虫文化（5分钟×1,000 XG），并删除网上平台。 </li><li> 19卷丙氨酸解悬浮颗粒（1毫升包装红细胞至19 ml丙氨酸解）。孵育15分钟，在RT。 </li><li>旋转5分钟×1,000×g的。吸去上清液。在RPMI-0一次清洗。吸上清，重悬在RPMI-A和红细胞压积调整至约3％。 G作为瓶中，回归到37℃。 注意:对于共培养实验，需要5％滋养体最小原虫，用10％的寄生虫血症认为是最优的。 </li></ol> 5，寄生虫和RBC准备共培养实验<ol><li>使用每个PBMC 3 PfRBC的共培养实验的比率以诱导炎症反应。计算总PfRBC，通过使薄的血涂片如3.5中所述，和血细胞比容通过使用血细胞计数器计数寄生虫培养的红细胞的数目每ml量化寄生虫血症。 PfRBC =％原虫数×总RBC /毫升×毫升文化。例如:在10×10 6的RBC / ml和10％的寄生虫血症10毫升培养物是等于0.1×10 6 /毫升×10毫升= 10×10 6 PfRBC。 </li><li>旋转5分钟寄生虫文化在1000 XG（RT）。吸出培养基，并以每毫升6×10 6 PfRBC在RPMI-S +（500毫升的RPMI-1640悬浮补充有L-谷氨酰胺和HEPES，10％热灭活的FBS，1.5 ml庆大霉素，5毫升的100mM丙酮酸钠，5毫升10毫MEM非必需氨基酸，加入5ml的5mMβ巯基乙醇的）。 </li><li>采取控制血压（未感染RBC从用于维护寄生虫文化同一供体），计算出每毫升相同数量的红细胞的寄生虫文化红细胞压积，重悬在RPMI-S +。 </li></ol> 6.分离人外周血单个核细胞（PBMC） <ol><li>收集来自慢性HIV（+）供体和艾滋病病毒的肝素钠管静脉血（绿顶）（ - ）的控制。不使用EDTA作为抗凝血剂如EDTA的钙螯合作用影响细胞功能。平均预期每10毫升血液之间5-10×10 6个PBMC。对于一个典型的实验收集30毫升的血液从每个捐赠者。血容量将需要根据实验要求调整。 </li><li>尽快绝对内收集血液一小时，取出的血液从管和地方变成塑料50ml管中。特别是单核细胞将被激活，并坚持玻璃，所以重要的是，如果玻璃血液收集管用于细胞被尽可能快地除去。 </li><li>旋血液在1000×g离心15分钟，并收集血浆（这可用于（如果需要）其它研究 - 如果不是这个步骤可以被跳过）。稀释血液中的冷DPBS等体积。 </li><li>放置15个ml的RT聚蔗糖的到50毫升管中。慢慢地，用无菌塑料移液管，层血液/ DPBS溶液在聚蔗糖，没有任何混合。最多25毫升血液/ DPBS解决方案可分层置于每个15毫升聚蔗糖的。 </li><li>旋梯度在室温30分钟，在600×g下。确保"不刹车"设置为开。 </li><li>一旦细胞纺丝，寻找两相之间的界面。这正是PBMC是。使用无菌塑料移液管从界面收集外周血单个核细胞（见图1）。受红细胞减少污染。 </li><li>将收集到的细胞在50毫升管，没有每管不少于20毫升。顶管可达50毫升无菌冷DPBS。 </li><li>旋转细胞10分钟，在300×G 4 摄氏度 。 </li><li>弃上清，重悬沉淀在5毫升无菌冷DPBS，集中来自同一供体的所有单元格成一管。补足至50ml无菌DPBS，并重复洗涤步骤2次以上。 </li><li>悬浮细胞在10ml的RPMI-S +介质。 </li><li>删除20微升等份，并与20微升台盼蓝的混合。移取10微升的血球计数和活细胞（即未采取了蓝染的细胞 - 所有的蓝色细胞都死了）。使用血细胞计数器来计算在溶液中细胞数量如下。 注:血细胞计数有规定的尺寸，使覆盖的行区域被称为一格。 <ol><li>确保血细胞计数板是干净的。将盖玻片在countiNG区域。负载10微升细胞溶液通过将移液管尖成V形井之一。盖玻片下的面积将通过毛细作用填充。 </li><li>放置装入血球计在显微镜下。该血球的满格包含1毫米2各9个方格。计算每个大型广场内的所有小区。如果有太多的细胞存在，稀释液进一步各显神通。 </li><li>计算细胞浓度如下:总细胞/ ml =（正方形的总细胞计数/＃）×稀释系数x万个细胞/ ml，20×10000个细胞/毫升= 20，例如，（500个细胞/ 5正方形）×稀释因子×10 6细胞总数。 </li></ol></li><li>调整培养基至10×10 6每毫升（RPMI-S +培养基）的最终浓度。 </li></ol> 7.疟疾/ HIV合并感染文化<ol><li>板100微升分离的PBMC和400微升的RPMI-S +培养基到24孔板（每孔1×10 6个PBMC）。保障这口井都设置了一式三份:3井未受感染的红细胞，3井PfRBC，3口井中，3口井PMA /离子霉素。这既是对HIV（+）和HIV（ - ）样本。 </li><li>对于PfRBC井，将3×10 6 PfRBC在每个孔（500微升寄生虫文化在5.2制备的）。 </li><li>对于未感染的红细胞的孔，将500微升在每个孔中制备5.3未感染的红细胞培养物。 </li><li>为介质的井​​，将500微升的RPMI-S +培养基在每个孔中。 </li><li>对于PMA /离子霉素井，准备PMA /离子霉素溶液（2.5微克/毫升，250微克/毫升分别）。放置将500μl此溶液在各孔中。 注意:作为T细胞的细胞因子分泌有效刺激物，PMA和离子霉素用作阳性对照，以确保细胞是功能性。 </li><li>将板在37℃，5％ 二氧化碳培养箱。 </li><li>可选:使用过量的细胞表型分析，使用流式细胞仪或存储在RN对于未来的mRNA表达分析的稳定的解决方案。 </li></ol> 8.疟疾的免疫反应检测注意:执行共培养实验，只要4天。在此期间，则无需更换培养基。的最佳时间点，将取决于所感兴趣的细胞类型和问的问题。 12-48小时的潜伏期是最佳的单核细胞反应PfRBCs，而淋巴细胞反应均在72-96小时最佳观测。的时间点需要根据实验问题进行优化。较短（2-4小时），可以使用如果完好PfRBC和PBMC中的相互作用是感兴趣。 <ol><li>喷丝板在300×g离心3分钟以沉淀细胞。收集700μl的培养物上清液从每孔中。 </li><li>旋转上清在1000 XG 5分钟，以清除任何杂物。 </li><li>等分澄清的上清液按需要，标签，并冷冻在-20℃以下，直至分泌因子分析是成为PERFORmed指。 </li><li>通过ELISA或珠阵列（按照制造商的建议方案）分析细胞因子/趋化因子的响应。 </li><li>收集残留在板成RNA稳定溶液的细胞和通过定量实时PCR 14-16利用用于mRNA表达分析。 </li></ol> 9.细胞内流式细胞特定小区Ccytokine响应使用外周血单个核细胞共培养P.恶性疟原虫感染的红细胞注意:如上所述共培养的长度将依赖于感兴趣的细胞类型。如果有兴趣的单核细胞反应PfRBC更短的潜伏期是必要的。时间将更长先天淋巴细胞应答γδT细胞，NK细胞，NKT细胞），和较长的仍然为CD4和CD8 T细胞。优化是必需的。 <ol><li> 6-8小时，在分析之前加入1微升的1000倍布雷菲德菌素A到所有的孔中。返回板块37℃培养箱。经过6-8小时incub通货膨胀，放置板置于冰上15分钟。 </li><li>删除使用轰轰烈烈的移液从孔中的细胞，并将它们放置标记microcentifuge管。刮可能需要以除去单核细胞。 </li><li>自旋细胞5分钟以1000×克。除去上清液。悬浮细胞在500μl流式细胞仪缓冲液（1×DPBS，2％热灭活的FBS，0.02％叠氮化钠）。 </li><li>除法细胞上，使得每个样品具有:一管对全抗体染色（240μL），和一个管荧光灯减一（FMO）对照抗体染色（240μL）。游泳池的少量每个样品（20微升）的未染色的流式细胞仪控制（这将在流式细胞仪设置流量使用）的。 </li><li>自旋细胞5分钟，在500×g下。除去上清液。 </li><li>孵育在50μl流式细胞仪缓冲液15分钟，细胞未缀合的抗人CD16 / CD32（5微克/毫升） 在 4℃阻断Fc受体。 </li><li>加入1毫升流式细胞仪缓冲液中向每个管。自旋CELLS 5分钟在500×g。除去上清液。 </li><li>悬浮细胞在50μl流式细胞仪缓冲液中的荧光团共轭抗体所需的细胞表面标志物的预滴定浓度。被染色所有样品预混足够抗体溶液。孵育细胞在4℃下 20分钟。从light.Note保护:各抗体的量将必须被优化。我们经常染色的CD56，CD3，γδ，CD4，CD8，CD14。滴定体积为这些抗体示于表1。 </li><li>加入1毫升流式细胞仪缓冲液中向每个管。自旋细胞5分钟，在500×g下。除去上清液。 </li><li>加入100微升cytofix / cytoperm解决方案，以每管。在4℃孵育细胞20分钟避光。 </li><li>加入1毫升的渗透/洗涤缓冲液（提供高达10倍的浓缩-稀释使用DDH 2 O的1倍），以每管。自旋细胞5分钟，在500×g下。除去上清液。 </li><li>加入100μ升烫发/洗缓冲FMO对照抗体染色管和混合重悬细胞。 </li><li>加入100微升的渗透/洗涤缓冲液中的荧光团共轭抗体所需的细胞内标记物（即，细胞因子/趋化因子），以完全抗体染色管的预滴定浓度。 注:各抗体的量将必须被优化。我们经常染色用抗TNF和抗IFNγ抗体。滴定体积为这些抗体示于表1。 </li><li>育所有试管30分钟，在4℃。避光。 </li><li>加入1毫升的渗透/洗涤缓冲液到每个管中。自旋细胞5分钟，在500×g下。除去上清液。 </li><li>悬浮细胞在300微升流式细胞仪缓冲液用1％多聚甲醛。让我们坐了至少15分钟，以中和HIV。 </li><li>准备使用补偿珠，以用于补偿建立在流式细胞仪单一抗体染色的样品。补偿的类型珠将取决于使用的抗体（即，抗小鼠Ig，抗大鼠/仓鼠Ig）的。涡珠。加入一滴每抗体珠。添加抗体等量作为用于染色。加入200μl流式细胞术缓冲区。这些都是现在可以使用。没有必要以洗涤珠。 </li><li>采集样本的流动，尽快仪和在24小时内获得最佳效果。串联染料易于分解与存储，所以我们建议立即采集如果可能的话。 </li></ol>

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The authors have nothing to disclose.

我们的协议进行了优化，以最真实地研究艾滋病疟疾双重感染体外 。首先，新鲜的人红细胞和血清所需疟原虫培养。这是至关重要的，以获得疟原虫的健康人群。寄生虫溶胞产物不能被取代的活寄生虫如细胞因子的产生是更快速和强烈的使用活P.时疟原虫感染的红细胞（PfRBC）17,18。此外，活化像NK细胞的细胞类型，需要整个PfRBCs，并且不与寄生虫溶胞产物有效地工作?...

合并感染，感染多个并发感染，是在自然的环境中的常态。共感染可以对疾病的病理和在每个感染的临床管理有重要影响。在共感染，疫苗和药物功效，以及诊断测试的范围内，可负面影响（在1中综述）。然而，尽管它的重要性，大多数病原体的研究只考虑单一的感染。 疟疾和HIV-1（艾滋病病毒）是全球领先的发病率和死亡率的原因。疟疾和艾滋病毒流行地区有着广泛的地理重叠，把数以百万计的人在共同感染的风险，因此，在风险更严重的临床疾病2 - 10。这两种疾病的负面互动。在艾滋病毒感染者，较高的HIV病毒载量和CD4 + T细胞计数暂时下降可在疟疾感染中可以看出，虽然疟疾寄生虫负担和临床和严重疟疾的风险是共同感染的个体2,3,5,7,8,10更高。由艾滋病病毒增加了疟疾严重性的机制尚不十分清楚，需要进一步调查。 在这里，我们描述了一个由疟疾和艾滋病病毒双重感染可在体外进行研究的方法，具体地讲，这种方法允许在感染艾滋病毒的情况下疟疾的特异性免疫反应的检查。我们的协议描述了从慢性感染艾滋病毒的捐助者，并在体外培养P.隔离使用新鲜分离的外周血单核细胞一个多功能的共培养体系（外周血单个核细胞） 疟原虫寄生红细胞（PfRBC）。 HIV抗逆转录病毒疗法对这些反应的影响，也可以使用前瞻性收集的PBMC来自HIV（+），供体前和治疗后研究。 我们已经使用这个系统来研究艾滋病病毒感染的影响疟疾特异性先天免疫反应11,12，并能够确定该疟疾特异性IFNγ和TNF响应在NK细胞受损，NKT细胞，γδŤ来自HIV（+），供体前后的HIV抗逆转录病毒治疗的细胞。此外，我们能够使用这个系统来确定单核细胞功能也受损HIV（+）捐助者，但恢复后的HIV抗逆转录病毒治疗。

<table><tbody><tr><td>alanine</td><td>Sigma</td><td>A7377</td><td></td></tr><tr><td>antibodies (see other table)</td><td></td><td></td><td></td></tr><tr><td>BD Cytofix/Cytoperm with BD GolgiPlug</td><td>BD</td><td>555028</td><td>includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer</td></tr><tr><td>BD Vacutainer ACD Solution A</td><td>BD</td><td>364506</td><td></td></tr><tr><td>BD Vacutainer Sodium Heparin</td><td>BD</td><td>17-1440-02</td><td></td></tr><tr><td>DPBS (no calcium, no magnesium)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F1051</td><td>heat inactivate before use</td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>gentamycin (10 mg/ml)</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>Hema3 Staining Set</td><td>Fisher</td><td>122-911</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma</td><td>H9636</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma</td><td>I3909</td><td></td></tr><tr><td>MEM non-essential amino acids (10 mM)</td><td>Gibco</td><td>11140</td><td></td></tr><tr><td>PMA</td><td>Sigma</td><td>P8139</td><td></td></tr><tr><td>RPMI-1640 powder</td><td>Life-Technologies</td><td>31800-022</td><td></td></tr><tr><td>RPMI-1640 with L-glutamine and HEPES</td><td>Thermo Scientific</td><td>SH30255.01</td><td></td></tr><tr><td>sodium bicarbonate (powder, cell culture)</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Gibco</td><td>11360</td><td></td></tr><tr><td>Tris (Trizma base)</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>Trizol</td><td>Ambion</td><td>15596018</td><td></td></tr><tr><td>Trypan Blue (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>BD CompBead</td><td>BD</td><td>552843, 552845</td><td>depends on antibodies used</td></tr><tr><td>Parasite Gas Mixture</td><td>By special order</td><td></td><td>3% CO&lt;sub&gt;2&lt;/sub&gt;, 1% O&lt;sub&gt;2&lt;/sub&gt;, balance N&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 &amp;#956;m filter unit</td><td></td><td></td><td></td></tr><tr><td>Glass slides</td><td></td><td></td><td></td></tr><tr><td>T25 flasks</td><td></td><td></td><td></td></tr><tr><td>T75 flasks</td><td></td><td></td><td></td></tr><tr><td>50 ml tubes</td><td></td><td></td><td></td></tr><tr><td>24 well culture plates</td><td></td><td></td><td></td></tr><tr><td>unplugged Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>plugged Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>sterile transfer pipettes</td><td></td><td></td><td></td></tr><tr><td>hemocytometer</td><td></td><td></td><td></td></tr><tr><td>flow cytometer (3 laser)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibodies used for flow cytometry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-TNF</td><td>eBiosciences</td><td>551487</td><td>FITC (fluorophore)
2 &amp;#956;l</td></tr><tr><td>Anti-IFN&amp;#947;</td><td>Biolegend</td><td>506507</td><td>PE (fluorophore) 
20 &amp;#956;l</td></tr><tr><td>Anti-CD8</td><td>Biolegend</td><td>300914</td><td>PE-Cy7 (fluorophore) 
1 &amp;#956;l</td></tr><tr><td>Anti-CD14</td><td>Biolegend; BD Biosciences</td><td>325624; 551487</td><td>Biotin (fluorophore) 
0.5 &amp;#956;l
Streptavin PE-TR (fluorophore) 
0.1 &amp;#956;l</td></tr><tr><td>Anti-CD56</td><td>Biolegend</td><td>318322</td><td>PerCP/Cy5.5 (fluorophore) 
5 &amp;#956;l</td></tr><tr><td>Anti-&amp;#947;&amp;#948;</td><td>Biolegend</td><td>331212</td><td>APC (fluorophore)
5 &amp;#956;l</td></tr><tr><td>Anti-CD3</td><td>Biolegend</td><td>300426</td><td>APC-Cy7 (fluorophore) 
5 &amp;#956;l</td></tr><tr><td>Anti-CD4</td><td>Biolegend</td><td>317424</td><td>Pacific Blue (fluorophore)
1 &amp;#956;l</td></tr></tbody></table>

an in vitro model for measuring immune responses to malaria in the context of hiv co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

该图描绘了IFNγ的产生由NKT细胞（图2）的水平，使用CD56 + CD3 +γδ-门以获得NKT细胞群（数据未显示）。将细胞培养72小时之前，染色。一旦沾上，被收购的流量100000 CD3 +细胞计数仪获得NK，NKT和γδ细胞（感兴趣的细胞）足够大的人群。最低5600 NKT细胞都显示每个图上。以相同的方式，获得TNF产生（数据未显示）。该图清楚地表明，IFNγ的产生是在细胞下的HIV（+）相比，艾滋病人（ - ）?...

Watch this Scientific Journal Video about An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection at JoVE.com

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

an-vitro-model-for-measuring-immune-responses-to-malaria-context-hiv

Research

JoVE Journal

Immunology and Infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

10.2K 次观看.  University of Calgary. Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection. 

视频: An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

科研

免疫与感染

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

干细胞基工程免疫抗HIV感染的人性化小鼠模型

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

医学

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

体外 细胞毒性淋巴细胞杀伤疟 原虫感染红细胞的测定

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

Generation of an In Vitro Cell Culture Model of Malaria-HIV Co-Infection

Source: Finney, C. et al., <a href="https://app.jove.com/v/52969">An In Vitro&#160;Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates the generation of an in vitro malaria-HIV co-infection cell culture model. HIV-infected peripheral blood mononuclear cells co-cultured with Plasmodium falciparum parasitized erythrocytes potentially support each other&#39;s growth and provide insights into the immune responses and disease progression in HIV and malaria co-infections.

疟疾-HIV 共感染体外细胞培养模型的生成

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
CAUTION: Working with human blood ...

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在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫

评估的HIV-1感染的CD4的先天传感

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

干细胞基工程免疫抗HIV感染的人性化小鼠模型

使用磁共振成像实验性脑疟疾模型中的水肿发育和微血管病理学的体内追踪

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

体外细胞毒性淋巴细胞杀伤疟原虫感染红细胞的测定

疟疾-HIV 共感染体外细胞培养模型的生成

一个

摘要

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此视频中的章节

一个

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫

评估的HIV-1感染的CD4的先天传感

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

干细胞基工程免疫抗HIV感染的人性化小鼠模型

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

体外 细胞毒性淋巴细胞杀伤疟 原虫感染红细胞的测定

疟疾-HIV 共感染体外细胞培养模型的生成

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

使用磁共振成像实验性脑疟疾模型中的水肿发育和微血管病理学的体内追踪

体外细胞毒性淋巴细胞杀伤疟原虫感染红细胞的测定