アン<em>インビトロ</em> 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
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| alanine | Sigma | A7377 | |
| antibodies (see other table) | |||
| BD Cytofix/Cytoperm with BD GolgiPlug | BD | 555028 | includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer |
| BD Vacutainer ACD Solution A | BD | 364506 | |
| BD Vacutainer Sodium Heparin | BD | 17-1440-02 | |
| DPBS (no calcium, no magnesium) | Corning | 21-031-CV | |
| Fetal Bovine Serum | Sigma | F1051 | heat inactivate before use |
| Ficoll-Paque PLUS | GE Healthcare | 17-1440-02 | |
| gentamycin (10mg/ml) | Gibco | 15710-064 | |
| Hema3 Staining Set | Fisher | 122-911 | |
| HEPES | Fisher | BP310-500 | |
| hypoxanthine | Sigma | H9636 | |
| Ionomycin | Sigma | I3909 | |
| MEM non-essential amino acids (10mM) | Gibco | 11140 | |
| PMA | Sigma | P8139 | |
| RPMI-1640 powder | Life-Technologies | 31800-022 | |
| RPMI-1640 with L-glutamine and HEPES | Thermo Scientific | SH30255.01 | |
| sodium bicarbonate (powder, cell culture) | Sigma | S5761 | |
| Sodium Pyruvate (100mM) | Gibco | 11360 | |
| Tris (Trizma base) | Sigma | T6066 | |
| Trizol | Ambion | 15596018 | |
| Trypan Blue (0.4%) | Gibco | 15250-061 | |
| BD CompBead | BD | 552843, 552845 | depends on antibodies used |
| Parasite Gas Mixture | By special order | 3% CO2, 1% O2, balance N2 |
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