Bir<em> İn Vitro</em> HIV Co-enfeksiyon Bağlamında Sıtma için Bağışıklık Yanıtları Ölçüm Modeli
概要
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-enfeksiyon, aynı anda birden çok enfeksiyonları ile enfeksiyon, doğal ortamlarında normdur. Co-enfeksiyon hastalığı patolojisi ve her enfeksiyonun klinik yönetimi üzerinde büyük bir etkiye sahip olabilir. Ko-enfeksiyon, aşı ve ilaç etkinliğinin yanı sıra, tanısal testlerin bağlamında, olumsuz etkilenebilir edilebilir (1 gözden). Ancak, onun önemine rağmen, patojen araştırma çoğunluğu sadece tek enfeksiyonları düşünmektedir.
Sıtma ve HIV-1 (HIV) küresel morbidite ve mortalite nedenleri lider vardır. 10 – sıtma ve HIV endemik Alanları ko-enfeksiyon riski milyonlarca insanı koyarak ve dolayısıyla daha ciddi klinik hastalık 2 için risk altında, geniş bir coğrafi örtüşme paylaşıyoruz. İki hastalık olumsuz etkileşim. HIV ile infekte olmuş kişiler, daha yüksek bir HIV viral yükü ve CD4 + T-hücresi sayımlarında geçici düşüşler olarak içinde ise, bir sıtma enfeksiyonu sırasında görülebilirsıtma paraziti yük ve klinik ve ciddi sıtma riski ko-enfekte olmuş bireylerin 2,3,5,7,8,10 daha yüksektir. HIV, sıtma şiddetini artıran hangi mekanizmalar tam olarak anlaşılamamıştır ve daha fazla incelemeyi gerektirdiği değildir.
Burada, in vitro incelenmiştir edilebilir sıtma ve HIV ko-enfeksiyonu ile bir yöntem açıklanmaktadır. Spesifik olarak, bu yöntem, HIV enfeksiyonu bağlamında sıtma spesifik immün yanıtların incelenmesi için izin verir. Bizim protokol kültürlenmiş P. kronik HIV ile enfekte donörlerden ve in vitro izole edilmiş taze izole edilmiş periferal kan mononükleer hücreleri kullanılarak çok yönlü bir ko-kültür sistemi (PBMC) tarif falsiparum eritrositler (PfRBC) parazitlenmiş. Bu tepkiler üzerine HIV antiretroviral tedavinin etkisi de HIV (+) bağış öncesi ve sonrası terapi prospektif olarak toplanan PBMC kullanılarak incelenebilir.
Biz HIV enfeksiyonu etkisini araştırmak için bu sistemi kullanmışsıtma özgü doğuştan gelen bağışıklık yanıtlarına 11,12 ve NK hücreleri engelli olduğu sıtma özgü IFNy ve TNF yanıtları belirlemek başardık, HIV (+) bağış öncesi ve sonrası HIV antiretroviral tedavi dan NKT hücreleri, γδ T hücreleri . Ayrıca, monositik işlevleri de HIV (+) vericilerden engelli olduğunu belirlemek, ama post-HIV antiretroviral tedavi kurtarmak için bu sistemi kullanmak mümkün.
Protocol
Representative Results
Discussion
Bizim protokol en gerçekçi in vitro HIV sıtma ko-enfeksiyon incelemek üzere optimize edilmiştir. İlk olarak, taze insan eritrositler ve serum sıtma paraziti kültürü için gereklidir. Bu sıtma parazitlerinin sağlıklı bir nüfus elde etmek için hayati önem taşımaktadır. Canlı P kullanırken sitokin üretimi daha hızlı ve yoğun olarak parazit lizatları canlı parazitler için ikame edilemez falciparum RBC (PfRBC) 17,18 enfekte. Buna ek olarak, NK hücreleri gibi…
開示
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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