<em> 체외</em> HIV 공동 감염의 맥락에서 말라리아에 면역 반응을 측정하기위한 모델
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
공동 감염, 복수의 동시 감염 감염은 자연 환경의 규범이다. 공동 감염 질환의 병리에 각각 감염의 임상 적 관리에 큰 영향을 미칠 수 있습니다. 공동 감염, 백신 및 약물 효능뿐만 아니라 진단 테스트의 맥락에서 부정적인 영향을받을 수있다 (1에서 검토). 그러나, 그것의 중요성에도 불구하고, 병원균 연구의 대부분은 하나의 감염을 고려한다.
말라리아 및 HIV-1 (HIV)는 전 세계적으로 이환율과 사망률의 원인을 선도하고 있습니다. 10 – 말라리아와 에이즈 endemicity 분야 공동 감염의 위험에 수백만의 사람들을 가하고 그 결과 더 심한 임상 질환 2의 위험, 넓은 지리적 중복을 공유 할 수 있습니다. 두 질환에 부정적인 상호 작용합니다. HIV 감염자 높은 HIV 바이러스 부하와 CD4 + T 세포 수가 일시적으로 감소하면서, 말라리아 감염 중에 알 수말라리아 기생충 부담과 임상 중증 말라리아의 위험이 공동 감염된 개인 2,3,5,7,8,10에서 더 높다. 에이즈 말라리아의 심각성을 증가하는 메커니즘은 완전히 이해하여 추가 조사를 보증하지 않습니다.
여기에서 우리는 체외에서 연구 할 수있는 말라리아 및 HIV 동시 감염하는 방법을 설명합니다. 특히,이 방법은 HIV 감염의 맥락에서 말라리아 특이 면역 반응의 시험을 허용한다. 우리 프로토콜 배양 P. HIV 만성 감염 기증자로부터 체외에서 격리 갓 절연 말초 혈 단핵 세포를 이용한 다기능 공 배양 시스템 (PBMC를)을 기술 원충은 적혈구 (PfRBC)를 기생. 이러한 응답에 HIV 항 바이러스 요법의 효과는 또한 HIV (+) 도너 전후 요법 전향 채취 한 PBMC를 사용하여 검사 할 수있다.
우리는 HIV 감염의 영향을 조사하기 위해,이 시스템을 사용한말라리아 관련 선천성 면역 반응에 대한 11, 12, 및 NK 세포에서 손상되는 말라리아 관련 IFNγ 및 TNF 반응을 확인할 수 있었다 HIV (+) 도너 전후 HIV 항 바이러스 요법에서 NKT 세포, γδ T 세포 . 또한, 우리는 단핵구 기능 또한 HIV (+) 기증자에 손상되는지 결정하지만, 이후 HIV에게 항 레트로 바이러스 치료를 복구하기 위해이 시스템을 사용할 수 있었다.
Protocol
Representative Results
Discussion
우리의 프로토콜은 가장 사실적으로 체외에서 HIV-말라리아 공동 감염을 연구하기 위해 최적화되었습니다. 첫째, 신선한 인간의 적혈구와 혈청은 말라리아 기생충 문화가 필요합니다. 이 말라리아 기생충의 건강한 인구를 얻기 위해 매우 중요합니다. 라이브 P. 사용시 사이토 카인 생성을 훨씬 더 빠르고 강력한 같이 기생충 해물 라이브 기생충 치환 될 수 없다 원충은 적혈구 …
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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