Summary

一个<em>体外</em>模型在HIV合并感染的背景下测量免疫应答疟疾

Published: October 06, 2015
doi:

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

合并感染,感染多个并发感染,是在自然的环境中的常态。共感染可以对疾病的病理和在每个感染的临床管理有重要影响。在共感染,疫苗和药物功效,以及诊断测试的范围内,可负面影响(在1中综述)。然而,尽管它的重要性,大多数病原体的研究只考虑单一的感染。

疟疾和HIV-1(艾滋病病毒)是全球领先的发病率和死亡率的原因。疟疾和艾滋病毒流行地区有着广泛的地理重叠,把数以百万计的人在共同感染的风险,因此,在风险更严重的临床疾病2 – 10。这两种疾病的负面互动。在艾滋病毒感染者,较高的HIV病毒载量和CD4 + T细胞计数暂时下降可在疟疾感染中可以看出,虽然疟疾寄生虫负担和临床和严重疟疾的风险是共同感染的个体2,3,5,7,8,10更高。由艾滋病病毒增加了疟疾严重性的机制尚不十分清楚,需要进一步调查。

在这里,我们描述了一个由疟疾和艾滋病病毒双重感染在体外进行研究的方法具体地讲,这种方法允许在感染艾滋病毒的情况下疟疾的特异性免疫反应的检查。我们的协议描述了从慢性感染艾滋病毒的捐助者,并在体外培养P.隔离使用新鲜分离的外周血单核细胞一个多功能的共培养体系(外周血单个核细胞) 疟原虫寄生红细胞(PfRBC)。 HIV抗逆转录病毒疗法对这些反应的影响,也可以使用前瞻性收集的PBMC来自HIV(+),供体前和治疗后研究。

我们已经使用这个系统来研究艾滋病病毒感染的影响疟疾特异性先天免疫反应11,12,并能够确定该疟疾特异性IFNγ和TNF响应在NK细胞受损,NKT细胞,γδŤ来自HIV(+),供体前后的HIV抗逆转录病毒治疗的细胞。此外,我们能够使用这个系统来确定单核细胞功能也受损HIV(+)捐助者,但恢复后的HIV抗逆转录病毒治疗。

Protocol

此协议需要捐助者的血清和红细胞用于寄生虫文化的招募和HIV(+)和未感染的捐助者外周血单个核细胞分离。机构审查委员会必须批准所有研究和所有捐助者必须提供之前抽血知情同意书。 警告:人类血液样本和人类疟原虫工作需要的预防措施。总是穿着白大褂,手套和工作在2级生物安全柜。在意外经皮接触到人类疟疾的情况下,报告给健康和安全的预防性治疗。额外的安…

Representative Results

该图描绘了IFNγ的产生由NKT细胞(图2)的水平,使用CD56 + CD3 +γδ-门以获得NKT细胞群(数据未显示)。将细胞培养72小时之前,染色。一旦沾上,被收购的流量100000 CD3 +细胞计数仪获得NK,NKT和γδ细胞(感兴趣的细胞)足够大的人群。最低5600 NKT细胞都显示每个图上。以相同的方式,获得TNF产生(数据未显示)。该图清楚地表明,IFNγ的产生是在细胞下的HIV(+)相比,艾滋病人( – )?…

Discussion

我们的协议进行了优化,以最真实地研究艾滋病疟疾双重感染体外 。首先,新鲜的人红细胞和血清所需疟原虫培养。这是至关重要的,以获得疟原虫的健康人群。寄生虫溶胞产物不能被取代的活寄生虫如细胞因子的产生是更快速和强烈的使用活P.疟原虫感染的红细胞(PfRBC)17,18。此外,活化像NK细胞的细胞类型,需要整个PfRBCs,并且不与寄生虫溶胞产物有效地工作?…

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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Cite This Article
Finney, C., Serghides, L. An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection. J. Vis. Exp. (104), e52969, doi:10.3791/52969 (2015).

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