我们感谢HBEC - 5I细胞旧金山康达尔，技术转让办公室，乔治亚州亚特兰大疾病预防控制中心。这项工作是由威康信托基金会（4年博士助学金交流，并在基础生物医学科学高级研究金的JAR，授予编号084226）。

一般建议人类大脑微血管内皮细胞（HBEC 5I）文化已先前所描述的6，8，P. 9， 培养恶性疟原虫的寄生虫。 HBEC - 5i和P.恶性疟原虫文化应保持在无菌条件下在任何时候都。所有试剂应在37 ° C预热我们建议定期检查支原体污染的PCR（Minevera Biolabs公司，以下制造商的指示）10。该协议是在图1总结。 1。内皮细胞常规培养<ol><li>准备必要的试剂。 </li></ol><table border="1"><tbody><tr><td> 中等至准备 </td><td> 试剂 </td><td> 数量 </td></tr><tr><td> “DMEM培养液不完整” </td><td> DMEM - F12火腿</td><td> 500毫升</td></tr><tr><td> ＆NBSP; </td><td> L -谷氨酰胺200MM </td><td> 5毫升</td></tr><tr><td></td><td>青霉素/链霉素100X </td><td> 5毫升</td></tr><tr><td></td><td>氢氧化钠1M </td><td> 1.3毫升（调整pH至7.4） </td></tr><tr><td> “DMEM完全” </td><td> “DMEM培养液不完整” </td><td> 450毫升</td></tr><tr><td></td><td>胎儿牛血清热灭活</td><td> 50毫升</td></tr><tr><td></td><td>内皮细胞生长补充</td><td> 5毫升</td></tr></tbody></table> <ol start="2"><li>在通风25厘米2瓶用10ml的DMEM完全培养基用5％的CO 2在37℃培养箱文化HBEC - 5I。 </li><li>当他们成为融合的传代细胞。由吸删除旧的介质和洗两次使用DMEM不完全介质或组织文化品位PBS（Ca 2 +和Mg 2 +的自由）预热至37 ° C。 </li><li>新增的预热尝试1毫升psin - EDTA（0.025％胰蛋白酶，EDTA的0.5MM），覆盖整个烧瓶，在37〜2分钟的漩涡° C。 </li><li>倒置显微镜下检查，已至少有90％的细胞分离。如果有必要，轻轻敲烧瓶底部打跑贴壁细胞。加入DMEM完全培养基15毫升锥形管的胰蛋白酶和转让细胞块10毫升。离心机在室温（RT）为300 克 4分钟。 </li><li>弃上清，悬浮颗粒的DMEM完全培养基用10ml。移液器向上和向下的解决方案，彻底悬浮细胞。 </li><li> 1或2毫升细胞悬液加入到一个新的培养瓶中，并添加新鲜培养基8毫升保持文化。每天倒置显微镜下评估文化的发展和改变介质的每2或3天，才变成黄色。 </li></ol> 2。准备选择的​​内皮细胞<ol><li>前两天的SelectIO的日子<html> N，添加纤维连接蛋白在无菌PBS（2微克/厘米2）在一个（或更多）60毫米培养皿稀释。 5至20分钟的菜在37 ° C，然后删除纤维连接蛋白的解决方案，一个月在4 ° C，可存储和重新使用一次。 </li><li>第1.3代细胞。到1.5。假设100％的融合细胞的分离和重新悬浮于10ml DMEM完全培养基（1.6节，与等量的培养基悬浮汇合是较低的，例如悬浮8毫升如果汇合率为80％），添加悬浮1.5毫升细胞纤维连接蛋白涂层的培养皿和另一个DMEM完全培养基1.5毫升。 </li><li>将在孵化器的种子培养皿。注：在理想的情况下，汇合将在选择的时间在90％左右，两天后。 </li><li> 可选 。要激活HBEC - 5I，添加在选择前的终浓度为50μg/ml24小时的TNF（肿瘤坏死因子）细胞因子。 </li></ol>e_title“&gt; 3。 恶性疟原虫常规培养<ol><li>准备必要的试剂（见表这里下）。人体红细胞（RBC）的分离通过全血（O组+），通过过滤器（见“疟疾研究方法”的出版，对于一般的疟疾寄生虫培养11）白细胞耗竭准备。两次离心5分钟为400 克和resuspending他们用10 ml的RPMI不全，洗涤红细胞。洗涤红细胞保持在4 ° C时，在不完整的中型50％血细胞比容。 </li></ol><table border="1"><tbody><tr><td> 中等至准备 </td><td> 试剂 </td><td> 数量 </td></tr><tr><td> “不完整的RPMI” </td><td> RPMI 1640（与碳酸氢钠） </td><td> 500毫升</td></tr><tr><td></td><td> HEPES 1M </td><td> 12.5毫升</td></tr><tr><td></td><td>葡萄糖20％ </td><td> 5毫升</td></tr><tr><td></TD&gt; <td> L -谷氨酰胺200MM </td><td> 5毫升</td></tr><tr><td></td><td>庆大霉素50mg/ml </td><td> 250μl </td></tr><tr><td></td><td>氢氧化钠1M </td><td> 0.7毫升（调节pH值至7.2） </td></tr><tr><td> “的RPMI完成” </td><td> “不完整的RPMI” </td><td> 450毫升</td></tr><tr><td></td><td>汇集人类（非）血清免疫</td><td> 50毫升</td></tr></tbody></table> <ol start="2"><li>文化体育恶性疟原虫感染的红细胞的RPMI完全培养基在2％的血细胞比容和孵化在37 ° C与1％O 2，CO 2，3％和96％N 2 。吉姆萨涂抹11日，以评估寄生虫（图2）发展阶段。 </li><li>定期（约每周一次）文化同步山梨醇治疗12。这一天之前选择检测，环形舞台文化的目的，在5％或更多的寄生虫血症（最好至少10％）。 </li></ol> 4。 P 的选择为cytoadhesion内皮细胞的恶性<ol><li>在一天的检测，寄生虫的文化应在HBEC - 5I文化色素滋养体阶段（ 图2）（理想10％的寄生虫血症），而应在50％至100％汇合（理想的90 ％）。每HBEC - 5I培养皿30μl包装细胞体积寄生虫文化需要。 </li><li>两次离心5分钟（500 克 ）寄生虫文化1.5毫升洗净寄生虫。弃上清，重悬新鲜的，温暖，DMEM不完全介质10毫升。重复洗第二次。悬浮与1.5毫升30μl包装不完整，用1％BSA的DMEM细胞体积。 </li><li>洗HBEC - 5I涂布培养皿两次吸气中期和不完整的DMEM加入3毫升。 </li><li>添加到HBEC - 5I菜和孵化的解决方案，寄生虫在37℃为75分钟彗星。两次重悬寄生虫在潜伏期（30和60分钟后）在四个方向，以及顺时针和反时针方向轻轻摇晃菜。 </li><li>孵育后，洗净的菜吸气中期的5倍，使用塑料巴斯德吸管加入3毫升温暖的DMEM不完全介质的，温和的摇摆。如果正在使用的许多菜肴，保持在一个温暖的表面，如装满了水在37大烧瓶° C。 </li><li>检查倒置显微镜下的菜。如果有很多未受感染的红细胞仍然可见，做如上所述的清洗。处理培养皿中非常小心，以避免任何污染风险。 </li><li>从盘中取出中期和温暖的RPMI完全培养基添加新鲜uRBC包装细胞体积40μl3毫升。 </li><li>放置在密闭的孵化室的菜，气3分钟，并将其放置在37℃过夜在一个孵化室。 </li><li>后的第二天，收获洗衣机用RPMI不完整的培养基，以类似的方式，如4.4节所述的寄生虫。但更积极。记p的所有介质（含悬浮红细胞）在15毫升锥形管。倒置显微镜下检查，所有RBC已经从菜中删除。 </li><li>颗粒的寄生虫，弃上清，悬浮5毫升的RPMI完全培养基和地方在正常培养瓶的混合物。 </li></ol> 5。代表性的成果未选中的寄生虫显示一个有约束力的HBEC - 5I（图3A）的低水平。因此，第一轮选拔后，极少数的寄生虫会有所收获，它可能需要长达一个月的培养，以达到足够的寄生虫血症的第二轮选择。每一轮选择后，越来越多的寄生虫内皮细胞结合的选择，可以在较短的时间间隔重复。经过4-5轮筛选，获得高约束力的寄生虫种群（图3）。 在我们的手中，HB3，HBEC结合HBEC - 5i或肿瘤坏死因子激活HBEC - 5I的SIM卡ILAR水平（数据未显示）。 这里描述的协议进行了测试，4 P。恶性疟原虫株：HB3，IT/FCR3，3D7和DD2（见表1）。只有后者被证明不能绑定到HBEC - 5I，即使经过5轮选拔。 HB3寄生虫也选择cytoadherence人体皮肤和肺微血管内皮细胞（HDMEC和HPMEC）。经过4轮选拔，使用方法这里描述的，高约束力的人口获得HDMEC和HPMEC（图4）。 <img alt="图1" src="/files/ftp_upload/3122/3122fig1.jpg" /> 图1。选拔过程的概述。 <img alt="图2" src="/files/ftp_upload/3122/3122fig2.jpg" /> 图2 P 的发展阶段。 恶性疟原虫感染的红细胞。吉姆萨涂片观察显微镜下的1000倍放大倍率。 <img alt="图3" src="/files/ftp_upload/3122/3122fig3.jpg" /> 图3。寄生虫HBEC - 5I结合的典型例子。 （一）蓝色层HBEC - 5I戊二醛固定，用姬姆萨染色，在显微镜下可视化1000X放大倍率。照片拍摄后uRBC和未绑定iRBC被洪水冲走。左侧面板显示一个单一的体育恶性 HB3（未选中）寄生虫势必HBEC - 5I。在右侧面板，HB3 HBEC寄生虫已被选定为5个回合。 （b）数据代表两个独立的实验，每次重复执行的平均水平。开往每内皮细胞的寄生虫数量计算。 <img alt="表1" src="/files/ftp_upload/3122/3122table1.jpg" /> 表1。摘要恶性疟原虫株已成功HBEC - 5I。注意结合 ，HB3也TNF激活HB选择选择EC - 5I。经过5轮筛选，DD2应变均没有约束力HBEC - 5I比未选中- DD2增加。 <img alt="图4" src="/files/ftp_upload/3122/3122fig4.jpg" /> 图4。 体育绑定恶性 HB3寄生虫真皮（HDMEC）和肺（HPMEC）内皮细胞前后4轮的选择，。虽然培养液中HDMEC和HPMEC略有不同（见供应商的说明），选择的协议是与相同HBEC - 5I。 400X的放大倍率拍照留念。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM - F12火腿</td><td>西格玛</td><td> D6421 </td><td>对于DMEM完全培养基</td></tr><tr><td> L -谷氨酰胺200MM </td><td>GIBCO公司</td><td> 25030 </td><td>对于DMEM和RPMI完全培养基</td></tr><tr><td>青霉素/链霉素100X（10000单位/ ml和10mg/ml的） </td><td> ScienCell </td><td> 0503 </td><td>对于DMEM完全培养基</td></tr><tr><td>胎儿牛血清热灭活</td><td> ScienCell </td><td> 0025 </td><td>对于DMEM完全培养基</td></tr><tr><td>胰蛋白酶- EDTA（0.025％胰蛋白酶，EDTA的0.5MM） </td><td> ScienCell </td><td> 0103 </td><td></td></tr><tr><td>内皮细胞生长补充</td><td> ScienCell </td><td> 1052 </td><td>对于DMEM完全培养基</td></tr><tr><td>组织培养处理60毫米× 15毫米培养皿</td><td>屋宇署</td><td> 353002 </td><td></td></tr><tr><td>人纤维连接蛋白</td><td> Millipore公司</td><td> FC010 </td><td>使用2微克/厘米2 </td></tr><tr><td>肿瘤坏死因子</td><td>研发D系统</td><td> 210 - TA </td><td>可选的，使用50微克/毫升</td></tr><tr><td> RPMI 1640培养</td><td>龙沙</td><td> BE12 - 167F </td><td>对于RPMI完全培养基</td></tr><tr><td>庆大霉素50mg/ml </td><td>龙沙</td><td> 17 - 518Z </td><td>对于RPMI完全培养基</td></tr><tr><td> HEPES 1M </td><td>龙沙</td><td> BE17 - 737E </td><td>对于RPMI完全培养基</td></tr><tr><td> HDMEC </td><td> ScienCell </td><td> 2000 </td><td>主细胞株</td></tr><tr><td> HPMEC </td><td> ScienCell </td><td> 3000 </td><td>主细胞株</td></tr><tr><td> HBEC - 5I </td><td></td><td></td><td>获得旧金山康达尔（fcandal@cdc.gov） </td></tr></tbody></table> 表2。

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+potentiate+brain+endothelial+alterations+induced+by+Plasmodium+falciparum.">Platelets potentiate brain endothelial alterations induced by Plasmodium falciparum.</a> Infect. Immun. 74, 645-653 (2006).</li></ol>

我们什么都没有透露。

脑型疟疾的特点是体育封存恶性 iRBC大脑内的微血管2，3。然而， 在体外培养的P.恶性不佳cytoadhere HBEC - 5I，人类大脑微血管内皮细胞模型。在这里，我们开发了一个简单的检测，以丰富HBEC - 5I结合，更表现型“一样在体内”的人口。三出4 P。恶性疟原虫株成功地使用这种方法选择。此外，HB3也HDMEC和HPMEC选择，这表明，这个协议可以用于各种寄生虫和内皮细胞类型。 不同的假设可以解释的缺乏约束力DD2应变。最有可能的事实，这种寄生虫线knobless，它深刻地阻碍了cytoadherence 13，14 。 我们建议选择亲一起培养未选中的寄生虫塞斯提供比较控制。例如，这将使比较转录的约束性和非约束性的寄生虫，发现寄生虫配位候选人的希望。 TNF是在高级别脑型疟疾患者中发现一种细胞因子，并已显示诱导HBEC - 5I（ 克拉森等人，在准备和8，15，16）的表面蛋白的表达。在这种情况下，势必iRBC金额是在正常的HBEC - 5I相似，而激活HBEC - 5I。 这种“封存模式”也可以用来研究iRBC和内皮细胞之间的分子间相互作用，以及公认的反cytoadherence药物的效果。在这种情况下，我们建议在较小的井，如“8以及玻片”（BD 354628）或“CultureWell”（Sigma公司C7735 - 20EA），电镀HBEC - 5I。

selection of plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells

大多数人患疟疾死亡血阶段的恶性疟原虫的寄生虫引起的。脑型疟疾，最危及生命的并发症的疾病，特点是由恶性疟原虫感染的红血球细胞色素滋养体阶段大脑微血管 2-4（iRBC）的积累。这微血管阻塞（封存），导致酸中毒，缺氧和有害的炎性细胞因子（审查5 ）。封存还发现，在人体2，3最微血管组织。其中iRBC附着于血管壁的机制仍然知之甚少。 永生化人脑微血管内皮细胞株（HBEC 5I）已被用来作为一个在体外血脑屏障模型。然而， 恶性疟原虫 iRBC附加只有糟糕HBEC - 5I 在体外 ，不像密集sequestrat离子在脑疟疾病例发生。因此，我们开发了一个平移检测选择（丰富）各种体育为了获得高约束性寄生虫，更能代表体内发生的人口恶性疟原虫株粘附HBEC - 5I。 寄生虫文化的一个样品色素滋养体阶段（iRBC和未受感染的红细胞混合物）洗涤和孵育生长在培养皿上HBEC - 5I层。孵化后，菜是轻轻洗涤uRBC和未绑定iRBC。新鲜uRBC添加到少数iRBC HBEC - 5i和孵育过夜。裂殖阶段寄生虫爆裂，裂殖子reinvade红细胞和这些环阶段的寄生虫是收获的翌日。寄生虫培养，直到获得足够的材料（一般为2至4周），并可以进行新一轮的选择。根据不同的 P. 恶性疟原虫株，需要4至第7轮的选择，以获得一个人口大多数寄生虫绑定HBEC - 5I。几个星期后，正逐步失去约束力的表型，说明变异表面抗原基因表达的开关，从而需要保持经常选择HBEC - 5I的表型。 综上所述，我们开发了一种选择检测呈现P.恶性疟原虫的寄生虫更“脑型疟疾的粘合剂”表型。我们能够选择4 P. 3 恶性疟原虫株HBEC - 5I。此实验也成功地被用来选择对人体皮肤和肺血管内皮细胞结合的寄生虫。更重要的是，这种方法可以用来选择组织特异性寄生虫种群，以确定寄生虫配体结合脑血管内皮细胞的候选人。此外，该法可用于屏幕上公认的反封存药品7。

Watch this Scientific Journal Video about Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells at JoVE.com

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

一个<em&gt;在体外</em&gt;脑型疟疾封存模型描述<sup&gt; 1</sup&gt;。<em&gt;恶性疟原虫</em&gt;受感染的红细胞被选中人脑微血管内皮细胞结合。选定的寄生虫表现出不同的表型。选择过程中可以使用各种应用<em&gt; P。恶性疟原虫</em&gt;株和血管内皮细胞株。

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

selection-plasmodium-falciparum-parasites-for-cytoadhesion-to-human

Research

JoVE Journal

Immunology and Infection

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

18.9K 次观看.  University of Edinburgh. 一个在体外脑型疟疾封存模型描述 1。恶性疟原虫受感染的红细胞被选中人脑微血管内皮细胞结合。选定的寄生虫表现出不同的表型。选择过程中可以使用各种应用 P。恶性疟原虫株和血管内皮细胞株。

视频: Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

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

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

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

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

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

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

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

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. ...

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

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

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

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

分离<em&gt;恶性疟原虫</em&gt;晚期感染的红细胞由磁性装置

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

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

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 ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

恒流中原发性人类内皮细胞的肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

脑膜炎奈瑟菌 诱导多能干细胞来源的脑内皮细胞的感染

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

选择恶性疟原虫寄生虫对人脑血管内皮细胞