Um<em> In Vitro</em> Modelo para medição de respostas imunes a Malária no Contexto do HIV co-infecção
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
Co-infecção, a infecção com múltiplas infecções simultâneas, é a norma em ambientes naturais. A co-infecção pode ter um impacto importante na patologia da doença e na gestão clínica de cada infecção. No contexto da co-infecção, vacina e a eficácia do fármaco, bem como os testes de diagnóstico, podem ser influenciadas negativamente (revisto em 1). No entanto, apesar da sua importância, a maioria das pesquisas patógeno considera infecções única individuais.
Malária e HIV-1 (HIV) são as principais causas de morbidade e mortalidade em todo o mundo. Áreas de malária e HIV endemicidade compartilhar uma ampla sobreposição geográfica, colocando milhões de pessoas em risco de co-infecção e, conseqüentemente, em risco de doença clínica mais grave 2-10. As duas doenças interagir negativamente. Em indivíduos infectados pelo HIV, a carga viral mais elevados e diminuições temporárias em + contagem de células T CD4 pode ser visto durante uma infecção por malária, enquantoencargos parasita da malária e risco de malária clínica e grave são mais elevados em indivíduos co-infectados 2,3,5,7,8,10. Os mecanismos pelos quais o HIV aumenta a gravidade da malária não são totalmente compreendidas e garante uma investigação mais aprofundada.
Aqui, descrevemos um método pelo qual a malária e a co-infecção com HIV pode ser estudado in vitro. Mais especificamente, este método permite a análise das respostas imunes específicas para a malária no contexto da infecção pelo HIV. Nosso protocolo descreve um sistema versátil de co-cultura utilizando células mononucleares do sangue periférico isoladas recentemente (PBMC) isoladas a partir de dadores cronicamente infectadas pelo VIH e cultivados in vitro P. falciparum parasitados eritrócitos (PfRBC). O impacto da terapia anti-retroviral HIV sobre estas respostas também podem ser examinados usando PBMCs prospectivamente coletados de VIH (+) doadores pré e pós-tratamento.
Temos utilizado este sistema para investigar o impacto da infecção pelo HIVna resposta imune inata específicas para a malária 11,12, e foram capazes de determinar que IFN-y e TNF respostas específicas para a malária são prejudicados nas células NK, células NKT, γδ células T de HIV (+) doadores pré e pós-terapia anti-retroviral HIV . Além disso, fomos capazes de utilizar este sistema para determinar que funções monoc�icas também são prejudicados em HIV (+) doadores, mas recuperar pós-HIV terapia anti-retroviral.
Protocol
Representative Results
Discussion
Nosso protocolo foi optimizado, a fim de estudar mais realisticamente co-infecção com HIV-malária in vitro. Em primeiro lugar, os glóbulos vermelhos humanos frescos e soro são necessários para a cultura parasita da malária. Isso é vital para obter uma população saudável de parasitas da malária. Os lisados de parasitas não pode ser substituído por parasitas vivos como a produção de citoquinas é muito mais rápida e intensa quando se utiliza vivo P. RBC infectados falciparum</e…
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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