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

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

このプロトコルは、寄生虫培養に使用される血清およびRBCのためのドナーの補充を必要とし、PBMCの単離のためのHIV（+）と非感染ドナー。機関審査委員会は、すべての研究を承認する必要があり、すべてのドナーは、血液採取の前にインフォームドコンセントを提供する必要があります。 注意：ヒトの血液サンプルと人間のマラリア原虫での作業は、予防措置が必要です...

<ol>
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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=HIV+infection+deregulates+Tim-3+expression+on+innate+cells:+combination+antiretroviral+therapy+results+in+partial+restoration.">HIV infection deregulates Tim-3 expression on innate cells: combination antiretroviral therapy results in partial restoration.</a> Journal of acquired immune deficiency syndromes. 63 (2), 161-167 (2013).</li><li>Finney, C. A. 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我々のプロトコルは、最も現実的に、インビトロでの HIV-マラリア同時感染を研究するために最適化されています。まず、新鮮なヒト赤血球と血清はマラリア原虫の培養に必要とされます。これは、マラリア原虫の健常者集団を得ることが不可欠です。サイトカイン産生をはるかに迅速かつ強烈であるとしてライブPを使用したとき寄生虫溶解物は、ライブ寄生虫の代わりに使用?...

同時感染、複数の同時感染症の感染は、自然環境では標準です。同時感染は、疾患の病理学上の各感染の臨床管理に大きな影響を持つことができます。同時感染、ワクチン及び薬物の有効性、ならびに診断試験の文脈では、悪影響を受けることができる（1に概説）。しかし、その重要性にもかかわらず、病原体研究の大半は、単一の感染を考慮します。 マラリアおよびHIV-1（HIV）は世界的に罹患率と死亡率の原因をリードしています。 10 -マラリアやHIVの地方的流行の領域は同時感染のリスクが何百万人もの人々を入れ、その結果、より重度の臨床疾患2のリスクが、広い地理的なオーバーラップを共有しています。 2疾患は否定的に相互作用します。 HIVに感染した個体は、より高いHIVウイルス負荷およびCD4 + T細胞数の一時的な減少をしながら、マラリア感染の間に見ることができますマラリア原虫の負担と臨床および重症マラリアのリスクが同時感染した個体2,3,5,7,8,10に高くなっています。 HIVは、マラリアの重症度を増加するメカニズムは完全に理解し、さらなる調査を保証されていません。 ここでは、マラリア、HIV同時感染をインビトロで研究することができる方法を記載している。具体的には、この方法は、HIV感染の文脈におけるマラリア特異的免疫応答の検査が可能になります。我々のプロトコルは、培養されたP.慢性的にHIVに感染したドナーからインビトロで単離新たに単離した末梢血単核細胞を用いた汎用性の高い共培養系（のPBMC）について説明熱帯熱マラリア原虫は赤血球（PfRBC）を寄生しました。これらの応答のHIV抗レトロウイルス療法の影響はまた、HIV（+）ドナー前および治療後からプロスペクティブに収集したPBMCを使用して検査することができます。 我々は、HIV感染の影響を調査するためにこのシステムを使用していますマラリア固有の自然免疫応答の11,12、およびNK細胞で損なわれることマラリア固有のIFNγおよびTNF応答を決定することができました、HIV（+）ドナー前後のHIV抗レトロウイルス療法からNKT細胞、γδT細胞。さらに、当社は、単球の機能はまた、HIV（+）ドナーにおいて減損が、後のHIV抗レトロウイルス療法を回復していると判断する。このシステムを使用することができました。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>alanine</td>
			<td>Sigma</td>
			<td>A7377</td>
			<td></td>
		</tr>
		<tr>
			<td>antibodies (see other table)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm with BD GolgiPlug</td>
			<td>BD</td>
			<td>555028</td>
			<td>includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer</td>
		</tr>
		<tr>
			<td>BD Vacutainer ACD Solution A</td>
			<td>BD</td>
			<td>364506</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Sodium Heparin</td>
			<td>BD</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (no calcium, no magnesium)</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>heat inactivate before use</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamycin (10mg/ml)</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hema3 Staining Set</td>
			<td>Fisher</td>
			<td>122-911</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>hypoxanthine</td>
			<td>Sigma</td>
			<td>H9636</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I3909</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids (10mM)</td>
			<td>Gibco</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 powder</td>
			<td>Life-Technologies</td>
			<td>31800-022</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 with L-glutamine and HEPES</td>
			<td>Thermo Scientific</td>
			<td>SH30255.01</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate (powder, cell culture)</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100mM)</td>
			<td>Gibco</td>
			<td>11360</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (Trizma base)</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Ambion</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>BD CompBead</td>
			<td>BD</td>
			<td>552843, 552845</td>
			<td>depends on antibodies used</td>
		</tr>
		<tr>
			<td>Parasite Gas Mixture</td>
			<td>By special order</td>
			<td></td>
			<td>3% CO2, 1% O2, balance N2</td>
		</tr>
	</tbody>
</table>

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

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

グラフは、NKT細胞の集団を得るためにCD56 + CD3 +γδ-ゲートを使用して、NKT細胞からのIFNγ産生（ 図2）のレベルを示している（データは示さず）。細胞は染色前に72時間培養しました。一度染色100,000 CD3 +細胞は、NK、NKTおよびγδ細胞（目的の細胞）の十分な大きさの集団を得るために、フローサイトメーターで得ました。 5600 NKT細胞の最小値を各グラフに表示されます。 TNF産生は?...

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

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

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

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

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

Research

JoVE Journal

Immunology and Infection

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

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

のための免疫担当マウスにおける漿液性卵巣癌の同所性モデル in vivoで</em腫瘍免疫応答の>腫瘍イメージングとモニタリング

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

V3の人口ベースのシーケンシングを用いたHIV - 1トロピズムの遺伝子型推論

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Seven Steps to Stellate Cells

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

HIV - 1コアのin vitroでの脱殻で

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

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

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

不定冠詞<em&gt;のin vitro</em研究する&gt;共同感染モデル<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</emヒトの第一次単球由来の免疫細胞に&gt;-HIV-1の相互作用

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

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

使用肺炎球菌植民に対するプロバイオティクスの影響を調査<em&gt;体外</em&gt;接着アッセイ

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...