Name: evaluation of extracellular vesicle function during malaria infection
Uploaded: 2018-02-14T14:30:00.000+00:00
Duration: 13 min 45 s
Description: Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

这项研究由诺华医学和生物研究基金会 (PYM)、戈特弗里德和朱莉娅 Bangerter Rhyner 基金会 (兆瓦和 PYM) 和大学 (PYM) 的研究池等部分财政支助。额外赠款包括瑞士政府优秀奖学金为外国学者 (对 KAB 和 SM)。我们感谢伊莎贝尔 Fellay 和 Solange Kharoubi 赫斯的技术支持。

根据 Swissethics (swissethics.ch) 的指导原则, 从健康捐赠者的血液中获得人体红细胞。
注意:恶性疟原虫寄生虫培养 (3D7) 和 EV 生产以前是在 Mbagwu ( et al) 中描述的。11由于P. 恶性疟原虫是人类病原体, 请查阅当地的规章以进行处理。文化应该保持不育的整个时间。
1. 电动汽车的荧光标记
注: 以下程序利用标签技术稳定地将绿色荧光染料 (PKH67) 与大脂肪族尾部结合在 EV 膜的脂质区内。反应是在200µL 最终染色量, 其中包含20µM 最终浓度的 PKH67。在室温下执行所有步骤 (20-25 °c)
<ol><li>在离心管中, 将100µg 的电动车放在1毫升的 PBS 中。</li>
	<li>将电动汽车在离心管中以最大速度 (2万 x g) 离心, 以7分钟为颗粒。 注意: 由于 hemozoin 存在于电动车中, 因此应观察到一种红色深褐色颗粒。如果上清是红色的, 那么电动车就有裂解。</li>
	<li>离心后, 小心地吸上清液, 以避免去除任何电动车。 注: 在稀释剂 C 中 resuspending 电动车之前, 尽量减少剩余的缓冲量, 以提高染色的重现性和效率。</li>
	<li>并用重悬100µL 稀释剂 C (2X ev 悬浮) 中的 EV 颗粒, 并轻轻吸管确保完全分散。</li>
	<li>准备一个新的离心管, 并添加4µL 的 PKH67 乙醇染料溶液的100µL 稀释剂 c. 漩涡井混合。在染色前立即准备此2x 染料溶液 (40 µM)。 注意: 为了避免异质染色, 请勿将 PKH67 乙醇染料溶液直接添加到 EV 颗粒上。</li>
	<li>快速, 结合100µL 2X EV 悬浮 (步骤 1.4) 与100µL 2X 染料溶液 (步骤 1.5)。然后, 通过吹打的10倍来混合样品。</li>
	<li>孵化的 EV/染料悬浮从步骤1.6 为5分钟保护, 从光和混合在5分钟孵化期间涡流3-4 倍。 注: 孵化的 EV/染料悬浮较长时间提供没有好处, 因为染色是如此之快。</li>
	<li>通过添加等量 (200 µL) 的血清和孵育1分钟来阻止染色反应, 从而使过量染料具有约束力。 注: 在停止染色前, 用 2万 x g 离心法从电动车上消耗20分钟的血清。</li>
	<li>用 pbs 清洗3次, 在 2万 x g 处旋转5分钟并用重悬电动车在µL 的100个, 达到最终浓度1µg/µL。</li>
</ol>2. 用共焦显微镜可视化电动汽车的吸收
注意: 下面的协议描述了在玻璃盖玻片上生长的内皮细胞对 EV 内化的跟踪。内皮细胞是半永生化的人骨髓内皮细胞, 如12所述。
<ol><li>在无菌环境中工作, 在室温下 (RT), 涂盖玻片0.01% 多聚 l-赖氨酸, 10 分钟以上。</li>
	<li>吸入聚 l-赖氨酸溶液, 使盖玻片完全干燥。</li>
	<li>使用台盼蓝排除和 hemocytometer 进行可行的内皮细胞计数。</li>
	<li>将 1 x 105内皮细胞移植到1毫升完整的内皮细胞生长培养基中, 其中含有补充剂与聚 l-赖氨酸涂层盖玻片, 并让细胞生长成单层。 注意: 培养基中含有生长因子, 允许细胞形成单层, 形成紧密的连接。</li>
	<li>一旦细胞形成单层, 仔细吸入培养基, 并添加1毫升的新鲜培养基来洗涤细胞。再重复一次洗涤。添加50µg PKH67-labeled 电动车和孵育4小时。 注意: 为了找到最佳的摄取时间点, 可以执行一个时间过程。</li>
	<li>4小时后, 吸入培养基, 用 PBS 冲洗细胞3次以去除多余和未绑定的电动车。将3% 多聚甲醛溶液的细胞固定在 PBS 中15分钟。 注: 甲醇可在固定过程中扰乱肌动蛋白;因此, 最好避免含有护或其他溶剂型护的甲醇。建议使用无甲醇甲醛作为固定剂。</li>
	<li>用 PBS 冲洗细胞3次。仔细添加250µL 0.1% 海卫 X-100 在 PBS permeabilize 细胞。在 RT 孵育5分钟。</li>
	<li>用 PBS 洗3次。在 RT 中添加400个阻塞缓冲区 (PBS 中的 5% BSA) µL, 以阻止非特定绑定。</li>
	<li>用 PBS 洗一次细胞。在200µL PBS/1%bsa 每套µL 中稀释5的罗丹明溶液, 以制备染色溶液。吸管200µL 的染色溶液在盖玻片和孵化20分钟的 RT。</li>
	<li>用 PBS 洗3次。Counterstain DNA 为三十年代与赫斯特33342在最后集中2µg 或 mL 在200µL PBS。</li>
	<li>通过添加400µL 的 PBS 来洗涤2x 的盖玻片。小心地抓住盖玻片与镊子, 并倒置它在一滴 antifade 安装介质上的玻璃幻灯片。用纸巾轻轻地取出多余的介质, 并用指甲油封住两侧。</li>
	<li>将受保护的幻灯片存储在4摄氏度的光线下。</li>
	<li>用共焦显微镜在激光激发405、488和 543 nm 和63X、1.3 NA 油目标上可视化细胞, 其荧光量为 450-470 nm (蓝色)、505-530 nm (绿色) 和 620 nm。 注意: 典型的 F 肌动蛋白染色结果显示在图 1中。</li>
</ol>3. 内皮细胞通透性
注: 通过测量内皮细胞单层中罗丹明 B 异硫氰酸酯-葡聚糖 (平均兆瓦 7万) 的转移, 评估内皮细胞通透性。葡聚糖为研究血管通透性提供了极好的工具。
<ol><li>使用0.4% 台盼蓝排斥和 hemocytometer 计数可行的内皮细胞。</li>
	<li>将内皮细胞密度为 0.6 x 105细胞以汇合24井组织培养插入物 (0.4 µm 孔径), 不受干扰地留在至少5天内形成分化的单分子膜。每隔一天刷新一次介质。</li>
	<li>一旦细胞达到单层, 载入电动车 (50 µg 在1毫升) 在上部室。将罗丹明-葡聚糖添加到顶部井中, 最终浓度为20毫克/毫升。孵化细胞在37°c 与 5% CO2。</li>
	<li>通过测量40µL 中整除数的荧光辐射, 监测荧光葡聚糖在井底的转移。设置多标签微板块读取器544毫微米励磁和 590 nm 发射测量。 注: 通过与库存溶液建立的校准曲线, 可以量化扩散葡聚糖的数量。此外, 通过在时间课程中去除外腔介质的小样本 (图 2), 可以进行动力学实验。即使没有任何治疗, 葡聚糖也会通过细胞单层扩散。</li>
</ol>4. 确定嘌呤霉素灵敏度
注意: 在传感细胞系与慢病毒北疆之前, 确定某一特定细胞系的药物的杀死曲线是很重要的。为了确定杀死所有细胞所需的药物浓度的最小量, 用所选药物的增量剂量进行剂量反应实验。由于每个哺乳动物细胞系都有不同的灵敏度, 在试验之前, 应根据以下详细情况确定抗生素的最佳浓度。
<ol><li>使用 hemocytometer 计数单元格。并用重悬内皮细胞浓度为 1 x10 5 细胞/毫升, 在完全内皮细胞生长培养基中含有补充剂组合物。</li>
	<li>板 1 x 104细胞成96井板在最后容量100µL 内皮细胞生长培养基。</li>
	<li>添加100µL 含有抗生素的培养基, 以获得 0.1-10 µg/毫升的浓度 (请参见图 3)。</li>
	<li>使用20X 目标, 每2天在光显微镜下检查细胞的生存能力。培养细胞10-14 天, 并根据细胞生长, 每2-3 天更换含有嘌呤霉素的培养基。</li>
	<li>在标准培养条件下, 将10µL/井 MTS 试剂添加到每个井中, 混合, 并孵育4小时37摄氏度。</li>
	<li>用 490 nm (图 3) 在一个振动筛上简单地摇动盘子, 用平板阅读器测量处理过的和未经治疗的细胞的吸光度。</li>
</ol>5. 慢病毒载体载体对内皮细胞的转导
<ol><li>板 1 x 105内皮细胞在1毫升完全培养基前的前一天转导。当细胞达到50-80% 融合时进行转导。</li>
	<li>在冰上解冻慢病毒北疆, 使整除数的病毒储存避免冻融循环。三十年代在 200 x g 的材料上旋转, 在管子打开之前, 避免溢出。</li>
	<li>在最终浓度为8µg/毫升的细胞中加入铵溴化物。 注: 铵溴有助于转导效率, 但在某些情况下, 它可能是有毒的细胞。因此, 在转导前执行一条杀死曲线来确定最佳浓度。</li>
	<li>轻轻地将盘子搅拌。增加适当数量的病毒微粒 (在 0.5-2 x 106微粒之间) 在适当的传染多样性 (语言) 和旋转板材轻轻地三十年代混合。</li>
	<li>离心机中的细胞-病毒粒子混合在45分钟的 300 x g, 以提高转导效率。在一夜之间孵化出37摄氏度的细胞病毒颗粒混合物。</li>
	<li>小心去除含有病毒的微粒介质, 并适当丢弃。用新鲜的, 预热的完整培养基替换。</li>
	<li>在第二天使用嘌呤霉素启动选定内容: 移除介质并用新鲜介质替换它, 其中包含嘌呤霉素的正确浓度(图 3)。</li>
	<li>收获细胞后嘌呤霉素选择和分离 rna 使用 rna 隔离套件后, 制造商的指示。</li>
	<li>使用反向转录套件对所选 miRNAs 进行 cDNA 合成 (请参阅材料表)。</li>
	<li>根据引用的协议13设置 qPCR 反应。</li>
</ol>

<ol>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WHO Malaria Report 2015. , (2015).</li><li>Miller, L. H., Baruch, D. I., Marsh, K., Doumbo, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenic+basis+of+malaria.">The pathogenic basis of malaria.</a> Nature. 415 (6872), 673-679 (2002).</li><li>Mantel, P. Y., Marti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+extracellular+vesicles+in+Plasmodium+and+other+protozoan+parasites.">The role of extracellular vesicles in Plasmodium and other protozoan parasites.</a> Cell Microbiol. 16 (3), 344-354 (2014).</li><li>Couper, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite-derived+plasma+microparticles+contribute+significantly+to+malaria+infection-induced+inflammation+through+potent+macrophage+stimulation.">Parasite-derived plasma microparticles contribute significantly to malaria infection-induced inflammation through potent macrophage stimulation.</a> PLoS Pathog. 6 (1), e1000744 (2010).</li><li>Schofield, L., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+processes+in+malaria+pathogenesis.">Immunological processes in malaria pathogenesis.</a> Nat Rev Immunol. 5 (9), 722-735 (2005).</li><li>Mantel, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria-infected+erythrocyte-derived+microvesicles+mediate+cellular+communication+within+the+parasite+population+and+with+the+host+immune+system.">Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system.</a> Cell Host Microbe. 13 (5), 521-534 (2013).</li><li>Regev-Rudzki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+communication+between+malaria-infected+red+blood+cells+via+exosome-like+vesicles.">Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles.</a> Cell. 153 (5), 1120-1133 (2013).</li><li>Szempruch, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles+from+Trypanosoma+brucei+Mediate+Virulence+Factor+Transfer+and+Cause+Host+Anemia.">Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia.</a> Cell. 164 (1-2), 246-257 (2016).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Mantel, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infected+erythrocyte-derived+extracellular+vesicles+alter+vascular+function+via+regulatory+Ago2-miRNA+complexes+in+malaria.">Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria.</a> Nat Commun. 7, 12727 (2016).</li><li>Mbagwu, S., Walch, M., Filgueira, L., Mantel, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+Characterization+of+Extracellular+Vesicles+in+Malaria.">Production and Characterization of Extracellular Vesicles in Malaria.</a> Methods Mol Biol. 1660, 377-388 (2017).</li><li>Schweitzer, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+newly+established+human+bone+marrow+endothelial+cell+line:+distinct+adhesive+properties+for+hematopoietic+progenitors+compared+with+human+umbilical+vein+endothelial+cells.">Characterization of a newly established human bone marrow endothelial cell line: distinct adhesive properties for hematopoietic progenitors compared with human umbilical vein endothelial cells.</a> Lab Invest. 76 (1), 25-36 (1997).</li><li>Wong, W., Farr, R., Joglekar, M., Januszewski, A., Hardikar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probe-based+Real-time+PCR+Approaches+for+Quantitative+Measurement+of+microRNAs.">Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs.</a> J Vis Exp. (98), (2015).</li><li>Mulcahy, L. A., Pink, R. C., Carter, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routes+and+mechanisms+of+extracellular+vesicle+uptake.">Routes and mechanisms of extracellular vesicle uptake.</a> J Extracell Vesicles. 3, (2014).</li><li>French, K. C., Antonyak, M. A., Cerione, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+docking+at+the+cellular+port:+Extracellular+vesicle+binding+and+uptake.">Extracellular vesicle docking at the cellular port: Extracellular vesicle binding and uptake.</a> Semin Cell Dev Biol. 67, 48-55 (2017).</li><li>Montecalvo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+transfer+of+functional+microRNAs+between+mouse+dendritic+cells+via+exosomes.">Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.</a> Blood. 119 (3), 756-766 (2012).</li><li>Idro, R., Jenkins, N. E., Newton, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis,+clinical+features,+and+neurological+outcome+of+cerebral+malaria.">Pathogenesis, clinical features, and neurological outcome of cerebral malaria.</a> Lancet Neurol. 4 (12), 827-840 (2005).</li><li>Jambou, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+adhesion+on+human+brain+microvascular+endothelial+cells+involves+transmigration-like+cup+formation+and+induces+opening+of+intercellular+junctions.">Plasmodium falciparum adhesion on human brain microvascular endothelial cells involves transmigration-like cup formation and induces opening of intercellular junctions.</a> PLoS Pathog. 6 (7), e1001021 (2010).</li><li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-secreted+miR-105+destroys+vascular+endothelial+barriers+to+promote+metastasis.">Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis.</a> Cancer Cell. 25 (4), 501-515 (2014).</li></ol>

作者没有什么可透露的。

一些寄生虫, 包括弓形虫、锥、利什曼原虫和滴虫, 触发受感染的宿主细胞释放电动车。根据病原体, 释放的电动车可以调节宿主免疫应答或调解寄生虫之间的细胞通讯6。然而, 几乎没有证据表明这些小囊泡是如何导致疟疾疾病的。在这里, 我们描述了几种方法来调查在疟原虫感染期间 EVs 的功能。例如, 通过用 PKH67 荧光染料对其进行标记, 可以有效地监测接收单元体内</e...

根据世界卫生组织的报告, 2015年全世界有2亿1200万起新的疟疾病例, 大约42.9万人死亡, 主要是五岁以下的儿童1。导致严重疾病的机制, 通常与血管功能障碍有关, 仍未定义为2。疟原虫-iRBCs 分泌小的双脂膜球体, 称为胞外囊泡 (EVs)。众所周知, 这些电动车与感染过程和宿主对感染的免疫反应有潜在的相关性;然而, 对这些小泡在疟疾感染期间的确切功能知之甚少3。他们有可能扮演两个重要的角色: 一方面, 他们可能通过激活巨噬细胞4,5来促进发病机制;另一方面, 他们可能会调解寄生虫之间的细胞通讯和寄生虫和主机6,7。事实上, 寄生虫可以通过电动车相互转移蛋白质或核酸。例如,锥寄生虫罗得西亚 EVs 可以转移毒力因子血清抗性相关 (), 并可针对其他 T . 寄生虫和宿主红细胞 8.此外,恶性疟原虫-iRBCs 通过在电动车内转移核酸来相互通信。这使得寄生虫能够优化和同步其生长。实际上, EVs 可能是 gametocyte 转换的主要调节器, 因此有助于对传输阶段7的调节。
电动车不仅能调节寄生虫, 而且还能调解寄生宿主之间的相互作用。我们最近发现, 电动车从 iRBCs 包含主机衍生的 microRNAs (miRNAs; 小 RNA 物种的范围内21-25 核苷酸9), 由人类内皮细胞占去。在电动车中的 miRNAs 形成一个稳定的复杂与 Ago2 (RNA 诱导沉默复合体的成员), 一旦交付给接受细胞, 能够明确地沉默基因表达和影响细胞的屏障属性10。为研究电动汽车的功能, 开发了标准协议。在这里, 我们首先描述一个协议, 允许电动车的荧光标记, 以调查他们的接收细胞的吸收。此外, 通过使用共焦显微镜, 可以跟踪 EV 的命运在细胞内。几种荧光染料可用于跟踪电动车。胺反应染料, 5 (和-6)-羧基双乙酸琥珀酸酯 (CFSE) 和 Calcein-在囊泡内成为荧光。我们更喜欢使用两亲的标签, PKH, 因为它给一个更明亮和更均匀的信号。此方法为了解 EVs 和接收单元之间的交互提供了重要信息。在某些情况下, 电动车绑定到细胞的表面, 一些囊泡迅速占去。在吸收后, 电动车将他们的货物运送到细胞中, 并在其中发挥调节作用。
在这里, 我们描述了一个协议, 以测量内皮细胞的屏障功能在体外通过量化的转移荧光葡聚糖通过细胞单层。更灵敏的示踪剂可以使用, 如核素标记。但是, 它们需要特殊的安全预防措施才能使用。还有其他的化验方法来测量体外屏障功能, 如 transendothelial 电阻 (志愿者), 它测量紧密接合的完整性。最后可视化 ZO-1, 一个紧密的连接蛋白, 通过免疫荧光允许评估紧密连接完整性以及10。由于 EVs 是复杂和异构实体, 包含若干具有潜在监管特性的货物, 因此过度表达 tshr 一个特定的 RNA 来研究它对接收单元的影响是有用的。因此, 我们还定义了一个协议, 其目的是生成稳定的单元格线, 表达感兴趣的 miRNA10。

<table><tbody><tr><td>PKH67 Green Fluorescent Cell Linker Mini Kit</td><td>Sigma-Aldrich</td><td>MINI67-1KT</td><td></td></tr><tr><td>Diluent C</td><td>Sigma-Aldrich</td><td>G8278</td><td></td></tr><tr><td>poly-L-lysine</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>PBS</td><td>ThermoFisher - Gibco</td><td>10010023</td><td></td></tr><tr><td>Phalloidin CF594</td><td>Biotium</td><td>#00045</td><td></td></tr><tr><td>Hoechst 33342</td><td>ThermoFisher</td><td>H3570</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>ThermoFisher</td><td>P36934</td><td></td></tr><tr><td>Rhodamine B isothiocyanate&amp;ndash;Dextran</td><td>ThermoFisher</td><td>R9379-250MG</td><td></td></tr><tr><td>Insert with PET membrane transparent Falcon&amp;nbsp;for plate 24 wells</td><td>Falcon</td><td>353095</td><td></td></tr><tr><td>Endothelial Cell Growth&amp;nbsp;Medium MV</td><td>Promocell</td><td>C-22020</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma-Aldrich</td><td>P9620-10ML</td><td></td></tr><tr><td>MTS Cell Proliferation Colorimetric Assay Kit</td><td>Biovision</td><td>K300-500</td><td></td></tr><tr><td>hexadimethrine bromide</td><td>Sigma-Aldrich</td><td>107689-10G</td><td></td></tr><tr><td>MISSION&amp;nbsp;Lenti microRNA, Human hsa-miR-451a</td><td>Sigma-Aldrich</td><td>HLMIR0583</td><td></td></tr><tr><td>MISSION Lenti microRNA, ath-miR416, Negative Control 1 Transduction Particles</td><td>Sigma-Aldrich</td><td>NCLMIR001</td><td></td></tr><tr><td>MISSION&amp;nbsp;Lenti microRNA, Human</td><td>Sigma-Aldrich</td><td>NCLMIR0001</td><td></td></tr><tr><td>Leica TCS SP5</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>miRNeasy mini Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>TaqMan MicroRNA Reverse Transcription Kit 1000 reactions</td><td>ThermoFisher</td><td>4366597</td><td></td></tr><tr><td>hsa-mir-451a&amp;nbsp;RT/750 PCR rxns</td><td>ThermoFisher</td><td>001141</td><td></td></tr><tr><td>U6 snRNA</td><td>ThermoFisher</td><td>001973</td><td></td></tr><tr><td>TaqMan Universal Master Mix II, with UNG</td><td>ThermoFisher</td><td>4440038</td><td></td></tr><tr><td>StepOnePlus Real-Time PCR System</td><td>ThermoFisher</td><td>4376600</td><td></td></tr></tbody></table>

evaluation of extracellular vesicle function during malaria infection

疟疾是由疟原虫寄生虫引起的危及生命的疾病, 而P. 恶性疟原虫是非洲大陆上最普遍的, 对全球大多数与疟疾有关的死亡负责。包括寄生虫在组织中的封存、血管功能障碍和炎症反应等几个因素影响了疟疾感染者的病情演变。恶性疟原虫-感染的红细胞 (iRBCs) 释放出小的胞外囊泡 (EVs), 其中含有不同种类的货物分子, 它介导寄生虫与宿主之间的致病机制和细胞间通讯。电动汽车是由它们调节其功能的细胞有效地占用的。在这里, 我们讨论的策略, 以解决电动车在寄生虫宿主互动的作用。首先, 我们描述了一种直接的方法来标记和跟踪的 EV 内化的内皮细胞, 使用绿色细胞链接染料。第二, 我们报告了一个简单的方法来测量渗透率跨内皮细胞单层, 使用荧光标记葡聚糖。最后, 我们展示了如何研究小的非编码 RNA 分子在内皮细胞功能中的作用。

在这里, 我们描述了研究电动汽车与宿主细胞相互作用的协议。荧光标记的 EVs 的摄取是通过共焦显微镜 (图 1) 来监测的。内皮细胞有效地占去了电动车, 但是利用 evs 的潜伏期可以优化以跟踪吸收。为了更好地定位电动汽车内部的细胞, 染色肌动蛋白与罗丹明。接下来, 我们使用一个过滤膜的顶端, 其中一层内皮细胞生长。罗丹明 B 异硫氰酸酯-葡聚糖?...

Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

Evaluation of Extracellular Vesicle Function During Malaria Infection

在这项工作中, 我们描述了研究由恶性疟原虫感染的红细胞释放出的细胞外囊 (EVs) 作用的协议。特别是, 我们关注的是电动车与内皮细胞的相互作用。

疟疾感染中胞外囊泡功能的评价

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

evaluation-of-extracellular-vesicle-function-during-malaria-infection

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.5K 次观看.  University of Fribourg. 在这项工作中, 我们描述了研究由恶性疟原虫感染的红细胞释放出的细胞外囊 (EVs) 作用的协议。特别是, 我们关注的是电动车与内皮细胞的相互作用。

视频: Evaluation of Extracellular Vesicle Function During Malaria Infection

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

科研

免疫与感染

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 ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

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 ...

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 ...

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.

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. ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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 ...

医学

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 ...

疟疾感染中胞外囊泡功能的评价

摘要

探索更多视频

此视频中的章节

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

一个

脑幽静的白细胞的分离与分析

高产净化

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

一个

方法调查小RNA和核糖体占用的调控作用

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

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

疟疾感染中胞外囊泡功能的评价

摘要

探索更多视频

此视频中的章节

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

一个

脑幽静的白细胞的分离与分析

高产净化

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

一个

方法调查小RNA和核糖体占用的调控作用

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

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

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