这项工作是由授予PRB-009，CS，博士生奖学金，LC，从国家的秘书CIENCIAŸTECNOLOGIA（SENACYT），巴拿马。

所有步骤的协议，除了离心，应进行发动机罩内保持无菌样品。 1。后期分离P.恶性疟原虫感染的红细胞疟原虫感染的红细胞的所有阶段的后期，可以用这种方法，分离自疟原虫色素，赋予对寄生虫的顺磁性，是一种常见的代谢物的属。甲高原虫（3-10％），建议在培养以获得更好的产率与该协议，虽然典型的培养将包含2-4％的血细胞比容（体积的红血细胞的百分比）和1-8％的原虫（百分比受感染的红血细胞）。寄生虫可以培养Trager的和Jansen 1所描述的。 <ol><li>一个简单的，多部分的设置必须进行组装，每个裂殖体的分离过程。对于这一点，LS列，磁MidiMACS的分离和MACS MultiStand美天旎生物技术（ 图1 </STRONG&gt;）。首先，将磁性分离器的金属底座，然后放置在隔板上的磁性列。 </li><li>在一个单独的管（50毫升conicals整个），将23.2毫升的RPMI 1640和750​​微升7.5％碳酸氢钠创建工作的解决方案。有额外的手头上的报废和收集管流过的寄生虫文化。 </li><li>的工作溶液中加入3毫升到柱中来平衡，并弃去流过成管。 </li><li>加入8毫升的P.恶性疟原虫文化到柱子上，确保，一旦工作解决方案的最后一部分已经被排挤出列的文化，接收管被替换为收集一个未绑定的文化开始恢复。这是必要的，以避免任何进一步稀释培养。 </li><li>一旦通过该柱的流动培养已经停止，加入1毫升的工作溶液推剩余的红细胞出列。 </li><li>清洗步骤：添加另一3毫升的工作溶液的柱并收集进入“丢弃”管中流动的液体，以避免稀释文化的其他管中被保存为流过。 </li><li>洗脱：一旦通过该柱的3毫升，分离后者从底座上，并且将其放置在15毫升管的顶部。 </li><li>同样，3毫升的工作溶液添加到柱中。这一步洗脱列的珠，被困在后期寄生虫感染的红细胞。 </li></ol> 注：在这一点上，并根据所需量的寄生虫，开始另一轮/ s的集合，明显的原始文化中的原虫的限制。该柱可以重复使用，只要流量保持稳定状态。 <ol start="9"><li>浓度为所收集的寄生虫：离心含有洗脱液在150×g离心5分钟，在室温下在15毫升的试管以形成一个黑暗的颗粒从寄生的红细胞。 </li><li>弃去上清，留出足够的液体，一个非常小的样本，并通过显微镜确定的浓度和总产率（见下文第2步）。 </li><li>在这一刻，如果完成令人满意的隔离和捕获后期寄生虫，孵育所收集的培养再次用新鲜培养基。 </li><li>稀释寄生在随后的实验中所需的体积和解决方案。 </li></ol> 2。纯度和产量的影响分析<ol><li>验证的浓度的寄生虫，通过以下方式获得使用血细胞计数仪，并在光学显微镜下计数。要计算，加10微升的混合收集的寄生虫和媒体的掩护下溜的血球计数仪。在四个象限中的血细胞计数仪计数的寄生虫数目，除以总数4。这个数字乘以10 4获得的浓度带毒rythrocytes每毫升。 </li></ol> 附注：）的浓度混合，将在由用户决定的时刻，再悬浮的沉淀，通过添加更多或更少的介质。一个建议的工作浓度是5.5×10 4 schizontes每微升（这通常是实现加入100-300μl到所收集的寄生虫）。如果此浓度的20微升被添加到最终体积为100μl的混合媒体和红细胞，原虫的约1％ - 在一个典型的4％的血细胞比容文化的方式获得。 b）点下的血细胞计数仪要快，因为作为样本箱内干燥的红细胞开始裂解。受感染的红细胞，这应该是大多数，会显示一个黑点内，他们从非疫区的区别。 <ol start="2"><li>执行的薄层Giemsa染色评估纯度的集合中，由固定在幻灯片非常迅速在10％甲醇中，干燥它们，和浸渍吨下摆的20％溶液的吉姆萨染色用蒸馏水稀释。 10分钟后，他们可以干燥空气，在显微镜下观察染色的幻灯片。一个原虫在70％以上应该是可以实现的。 </li></ol>

<ol>
	<li>Jensen, J. B., Trager, W. <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+in+culture:+use+of+outdated+erythrocytes+and+description+of+the+candle+jar+method.">Plasmodium falciparum in culture: use of outdated erythrocytes and description of the candle jar method.</a> J. Parasitology. 63 (5), 883-886 (1977).</li><li>Guzman, I. Y., Francis, S. E., Oksman, A., Smith, C. E., Duffin, K. L., Goldberg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+specificity+of+the+Plasmodium+falciparum+hemoglobin+degradation+pathway.">Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.</a> J. Clin. Invest. 93, 1602-1608 (1994).</li><li>Rosenthal, P. J., Meshnick, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemoglobin+catabolism+and+iron+utilization+by+malaria+parasites.">Hemoglobin catabolism and iron utilization by malaria parasites.</a> Mol. Biochem. Parasitol. 83 (2), 131-139 (1996).</li><li>Fitch, C. D., Chevli, R., Kanjananggulpan, P., Dutta, P., Chevli, K., Chou, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+ferriprotoporphyrin+IX+is+a+lytic+agent.">Intracellular ferriprotoporphyrin IX is a lytic agent.</a> Blood. 62 (6), 1165-1168 (1983).</li><li>Hebbel, R. P., Eaton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathobiology+of+heme+interaction+with+the+erythrocyte+membrane.">Pathobiology of heme interaction with the erythrocyte membrane.</a> Semin. Hematol. 26 (2), 136-149 (1989).</li><li>Egan, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemozoin+formation.">Haemozoin formation.</a> Mol. Biochem. Parasitol. 157 (2), 127-136 (2008).</li><li>Hempelmann, E., Marques, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+malaria+pigment+from+Plasmodium+falciparum.">Analysis of malaria pigment from Plasmodium falciparum.</a> J. Pharmacol. Toxicol. Methods. 32 (1), 25-30 (1994).</li><li>Fitch, C. D., Kanjananggulpan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+ferriprotoporphyrin+IX+in+malaria+pigment.">The state of ferriprotoporphyrin IX in malaria pigment.</a> J. Biol. Chem. 262 (32), 15552-15555 (1987).</li><li>Moore, L. R., Fujioka, H., Williams, P. S., Chalmers, J. J., Grimberg, B., Zimmerman, P. A., Zborowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemoglobin+degradation+in+malaria-infected+erythrocytes+determined+from+live+cell+magnetophoresis.">Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis.</a> FASEB J. 20 (6), 747-749 (2006).</li><li>Spadafora, C., Gerena, L., Kopydlowski, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+in+vitro+invasive+capabilities+of+Plasmodium+falciparum+schizonts+isolated+by+Percoll+gradient+or+using+magnetic+based+separation.">Comparison of the in vitro invasive capabilities of Plasmodium falciparum schizonts isolated by Percoll gradient or using magnetic based separation.</a> Malaria J. 10, 96 (2011).</li><li>Jackson, K. E., Spielmann, T., Hanssen, E., Adisa, A., Separovic, F., Dixon, M. W., Trenholme, K. R., Hawthorne, P. L., Gardiner, D. L., Gilberger, T., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+permeabilization+of+the+host+cell+membrane+of+Plasmodium+falciparum-infected+red+blood+cells+with+streptolysin+O+and+equinatoxin+II.">Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II.</a> Biochem. J. 403, 167-175 (2007).</li><li>Goodyer, I. D., Johnson, J., Eisenthal, R., Hayes, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+mature-stage+Plasmodium+falciparum+by+gelatine+flotation.">Purification of mature-stage Plasmodium falciparum by gelatine flotation.</a> Ann. Trop. Med. Parasitol. 88 (2), 209-211 (1994).</li><li>Pasvol, G., Wilson, R. J., Smalley, M. E., Brown, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+viable+schizont-infected+red+cells+of+Plasmodium+falciparum+from+human+blood.">Separation of viable schizont-infected red cells of Plasmodium falciparum from human blood.</a> Ann. Trop. Med. Parasitol. 72, 87-88 (1978).</li><li>Pertoft, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+cells+and+subcellular+particles+with+Percoll.">Fractionation of cells and subcellular particles with Percoll.</a> J. Biochem. Biophys. Methods. 44 (1-2), 1-30 (2000).</li><li>Paul, F., Roath, S., Melville, D., Warhurst, D. C., Osisanya, J. O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+malaria-infected+erythrocytes+from+whole+blood:+use+of+a+selective+high-gradient+magnetic+separation+technique.">Separation of malaria-infected erythrocytes from whole blood: use of a selective high-gradient magnetic separation technique.</a> The Lancet. 318, 70-71 (1981).</li><li>Trang, D. T., Huy, N. T., Kariu, T., Tajima, K., Kamei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+concentration+of+malarial+parasite-infected+red+blood+cells+and+removal+of+contaminating+white+blood+cells.">One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells.</a> Malar. J. 3, 7 (2004).</li><li>Nillni, E. A., Londner, M. V., Spira, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+separation+of+uninfected+erythrocytes+from+those+infected+with+Plasmodium+berghei+and+for+isolation+of+artificially+released+parasites.">A simple method for separation of uninfected erythrocytes from those infected with Plasmodium berghei and for isolation of artificially released parasites.</a> Z. Parasitenkd. 64, 279-284 (1981).</li></ol>

我们什么都没有透露。

在体外培养的疟疾寄生虫P.疟原虫表现出有限的原虫，超过一半的未感染的红血细胞，在最高的增殖培养点。对于大多数的研究实验中，它是理想的工作只与感染的红细胞。为此，一个分离的技术是必要的划分所述培养感染。有用的方法包括使用的链球菌溶血素O，以透性和lyze的未感染RBC 11和一系列利用两组不同密度的变化的差离心。这些方法的一些包括明胶浮选12?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> RPMI 1640的Hepes修改</td><td> Sigma-Aldrich公司</td><td> R4130 </td><td>补充有10％人血清，2％葡萄糖和0.2％的碳酸氢钠</td></tr><tr><td> MidiMACS分离</td><td> MACS美天旎生物技术公司</td><td> 130-042-302 </td><td></td></tr><tr><td> MACS MultiStand </td><td> MACS美天旎生物技术公司</td><td> 130-042-303 </td><td></td></tr><tr><td> LS列</td><td> MACS美天旎生物技术公司</td><td> 130-042-401 </td><td></td></tr><tr><td>血球</td><td> Grafco </td><td> Grafco纽鲍尔厅</td><td>可以发现，通过其他许多供应商</td></tr></tbody></table>

separation of plasmodium falciparum late stage-infected erythrocytes by magnetic means

与其他种类的疟原虫，P.恶性疟原虫可以在实验室中培养，这有利于研究1。虽然原虫能够达到≈40％的限制，研究者通常的比例保持在10％左右。在许多情况下，它是必要的隔离含寄生虫的红血细胞（红细胞）来自未感染的，以丰富的文化和继续进行，一个给定的实验。 当P.恶性疟原虫感染的红细胞，寄生虫降解和，馈送来自血红蛋白的2，3。然而，寄生虫必须处理的一个非常有毒的含铁血红素部分4，5。寄生虫逃避其毒性称为haemozoin 6，7成惰性液晶聚合物通过转化的血红素。此含铁分子被存储在其食物泡和在它的金属有一个从一个在血红素8中不同的氧化状态。血红素铁的铁状态ozoin赋予它在未受感染的红细胞没有一个顺磁性。作为入侵的寄生虫成熟，的haemozoin含量也增加9，赋予更顺磁性的 P上的最新的阶段恶性疟原虫红细胞内。 基于此顺磁性，最新的阶段P.恶性疟原虫感染的红血细胞可通过一列含有磁珠通过培养分离。这些珠粒成为磁性的磁体保持器时，被放置在含有它们的列。感染的红细胞，由于其顺磁性，然后将被媚惑​​列，而将包含流过，在大多数情况下，未感染的红细胞和那些含有早期阶段的寄生虫。 在这里，我们描述的方法，以丰富的人口后期寄生虫与的磁性列，保持着良好的寄生虫可行性10。在执行此过程，独立的文化可以返回一个孵化器，让剩余的寄生虫将继续增长。

在图2中，示出的磁性列通过培养之前，（A）和（B）后的程序。一到两个感染的红细胞通常出现在一个放大100倍的字段，如在图2中所示，在图2A中的感染的红细胞上，箭头指向。在一个典型的过程中，在5％的寄生虫血症（ 图2A）与培养开始时，此过程的性能，通常会产生红细胞与原虫的97％-100％（ 图2B）。?...

Watch this Scientific Journal Video about Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means at JoVE.com

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

顺磁性质的疟原虫色素是用来隔离后期阶段<em&gt;恶性疟原虫</em&gt;感染的红血细胞在培养皿中生长。该方法简单，快速，不影响后续的寄生虫的侵袭能力。

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 ...

separation-plasmodium-falciparum-late-stage-infected-erythrocytes

Research

JoVE Journal

Immunology and Infection

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

13.8K 次观看.  Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). 顺磁性质的疟原虫色素是用来隔离后期阶段恶性疟原虫感染的红血细胞在培养皿中生长。该方法简单，快速，不影响后续的寄生虫的侵袭能力。

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

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

前肿瘤反应性T细胞的体外扩增Bryostatin 1/Ionomycin手段和共同的γ链细胞因子配方

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

科研

免疫与感染

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

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

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

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

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 ...

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 ...

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. ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

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 ...

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 ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...