Name: an in vitro model for measuring immune responses to malaria in the context of hiv co-infection
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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.

<ol>
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The authors have nothing to disclose.

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 hücre tipleri aktivasyonu, tüm PfRBCs gerektirir ve parazit lizatları ile verimli bir şekilde çalışmaz. Bunun nedeni, PfRBC ve lökosit arasında doğrudan bir temas için ihtiyaç olabilir veya hücre yüzeyi reseptörleri 18 ile etkileşim parazit kaynaklı ligandların kararsız olmaları nedeniyle olabilir. Sıtma PfRBC kültürleri de iyi deney öncesinde (Protokol 4) senkronize olmalıdır. Senkronize PfRBCs deneysel verilerin tekrarlanabilirlik geliştirmek. Bu protokol, de rağmenscribes ko-kültür için trofozoit aşaması PfRBC kullanımı kolayca halka sahne PfRBC çalışma için modifiye edilebilir. İnsan lökosit yapay HIV ile enfekte olabilir ve bu yöntem, sıtma-HIV ko-enfeksiyonu araştırma 20 çalışmalarında kullanılmıştır. Bununla birlikte, bu, kronik HIV enfeksiyonundan sonuçlanan bağışıklık düzensizliği modeli etmez. Bu eş-kültür sistemine biz kronik HIV-1 ile enfekte olmuş insan katılımcı izole PBMC&#39;ler kullanın. Katılımcılar bir çalışma için gerekli olan zaman, dikkatle bu nüfusu seçmek önemlidir. İçerme ve dışlama kriterleri minimum değişkenliği ve maksimum tekrarlanabilirlik sağlamak için hayati öneme sahiptir. Bizim kriterleri, kronik HIV enfeksiyonu net bir tanımını dahil ve eşzamanlı enfeksiyon olan herkes dışlama (&gt; 50 hücre / mm 3 / yıl CD4 + T hücre sayısı düşüş ile,&gt; 1 yıl HIV bulaşmış). Bu elde edilen sonuçlar bu yüzden isnat edilebilir sağlamıştırKronik HIV enfeksiyonunun etkisi ve başka bir enfeksiyona lely. Biz sıtma için doğuştan gelen bağışıklık yanıtları ilgilenen beri Ayrıca, önceki sıtma enfeksiyonu vardı bağışçılar hariç. HIV ile enfekte olmayan kontrol aynı zamanda veri normalleştirme için izin vermek için her bir numune toplama noktasında, her HIV (+) donör için kullanılır. Bir girişim en azından yaş için kendi HIV (+) donör denetimleri maç için yapılmalıdır fakat tercihen de seks (her ne kadar önceki çalışmamızda 12 biz hücre alt veya kadın ve erkek arasındaki sitokin yanıtları önemli bir fark saptamadık HIV bulaşmamış katılımcı). Prospektif örnekleme her örnekleme zamanı noktası için aynı HIV ile enfekte olmayan kontrol sisteminin tasarlanmasını planlanmış ise faydalıdır. Yanıtlar PfRBCs sağlığı, senkronizasyon, parasitemia düzeyi ve PfRBCs ve olgunluk aşamasına derecelerine ve hematokrit düzeyi de dahil birçok faktörün nedeniyle deneme deney değişebilir. Özendeneyler arasında bu tutarlı gibi birçok tutmak için alınmalıdır. HIV ile enfekte olmayan kontrol normalleştirme bu değişkenler için bir muhasebe sağlar. Taze hücreler kullanılarak yapay sonuçlardan kaçınmak için her şeyden önemlidir. Donma ve çözülme örnekleri hücre canlılığı 21,22 üzerinde önemli bir etkiye sahip olabilir, sitokin üretimi 23-26 ve hücre yüzey fenotipik 27 işaretleme kalemler. Monositler özel ilgi konusudur, bu cam protokolü esnasında kullanılmaz önemlidir. Monositler cam ve diğer birçok plastik 28. yapışır. Bizim çalışma hücre popülasyonlarının gelen monosit yapışmasını ve nihai kaldırılmasını aza indirmek için boyunca polipropilen plastik pipetler, transfer pipet, ve tüpler kullanın. Tahlilinde flow sitometri kurarken, antikorlar herhangi bir kombinasyonu ilgi hücrelere bağlı olarak kullanılabilir. Biz IFNy ve TNF üretiminin özellikle ilgiNK hücreleri, NKT hücreleri ve γδ T hücrelerinden. Bu, bir antikor paneli tanımlar. Farklı bir kombinasyonu kullanılarak, ancak, her bir panel için antikor miktarları optimize etmek için önemlidir. Bu hatalı veri önlemek için hayati önem taşımaktadır. (-) Ve HIV (+), Şekil 2 HIV () Ayrıca, geçerliliğini değişkenliği önlemek ve sağlamak, FMOs her koşul (RBC, PfRBC, orta ve PMA / İyonomisin) ve katılımcı nüfus için plan sitokin düzeylerini değerlendirmek gerekmektedir. In vitro hücre sitokin üretimi Ölçüm genellikle hücrelerin boyamadan önce kültüre edilmiş, özellikle yüksek arka plan seviyelerinde sonuçlanır. Kültür koşulları arka plan düzeylerini etkileyen bu yana, uygun FMOs her bir numune için plan düzeyinde bilgi sağlamak. Hücrelerin Tedarik nedenle tüm yüzey lekeleri ancak hücre içi lekeleri içermeyen bizim FMOs tasarlanmış, sınırlı oldu. Bu çalışılan tüm farklı hücre tipleri ile ilgili sitokin arka plan seviyelerinde spesifik karşılaştırma sağlar.Biz de onların arka plan seviyelerini teyit etmek için yüzey belirteçlerinin her Deneme başına tek leke koştu. Açıklanan protokol ilgi hücrelere bağlı olarak saat veya gün içinde yanıt incelemek için kullanılabilecek bir çok yönlü bir biridir. Doğuştan gelen lemfositler optimal PfRBC sebep olduğu sitokin üretimi sağlamak için, 2 ya da 3 gün sonra çıkış akış sitometrisi ölçüldü. Monositler bakarken, daha önceki zaman noktaları (1 veya 2 gün) önerilir. Bu sistem, çıkarılan RNA olarak değerlendirilmesi gereken çok sayıda hücre türleri ve hücre yanıtlarının akışı ile ayırt edilmesi sitometrisi salgılama tepkileri hücre süpernatantlan içinde ölçülecek ve sentezleme profilleri ile karşılaştırın. Bundan başka, antikorların kullanımı belirli reseptörleri bloke eden ya da sitokinler daha mekanizmaları incelemek için, bu sistemde de kullanılabilir nötralize etmek. Başarıyla PfRBC kaynaklı IFNy yanıtları 12, IL-18 reseptörünü bulaştırmak için, IL-18 reseptör blokajını kullandık. Bu sistem, bir realist temsilHIV enfeksiyonu bağlamında sıtmaya karşı doğal immün yanıtların sayısını belirlemek için hangi ic yöntemi.

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.

<table><tbody><tr><td>alanine</td><td>Sigma</td><td>A7377</td><td></td></tr><tr><td>antibodies (see other table)</td><td></td><td></td><td></td></tr><tr><td>BD Cytofix/Cytoperm with BD GolgiPlug</td><td>BD</td><td>555028</td><td>includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer</td></tr><tr><td>BD Vacutainer ACD Solution A</td><td>BD</td><td>364506</td><td></td></tr><tr><td>BD Vacutainer Sodium Heparin</td><td>BD</td><td>17-1440-02</td><td></td></tr><tr><td>DPBS (no calcium, no magnesium)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F1051</td><td>heat inactivate before use</td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>gentamycin (10 mg/ml)</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>Hema3 Staining Set</td><td>Fisher</td><td>122-911</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma</td><td>H9636</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma</td><td>I3909</td><td></td></tr><tr><td>MEM non-essential amino acids (10 mM)</td><td>Gibco</td><td>11140</td><td></td></tr><tr><td>PMA</td><td>Sigma</td><td>P8139</td><td></td></tr><tr><td>RPMI-1640 powder</td><td>Life-Technologies</td><td>31800-022</td><td></td></tr><tr><td>RPMI-1640 with L-glutamine and HEPES</td><td>Thermo Scientific</td><td>SH30255.01</td><td></td></tr><tr><td>sodium bicarbonate (powder, cell culture)</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Gibco</td><td>11360</td><td></td></tr><tr><td>Tris (Trizma base)</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>Trizol</td><td>Ambion</td><td>15596018</td><td></td></tr><tr><td>Trypan Blue (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>BD CompBead</td><td>BD</td><td>552843, 552845</td><td>depends on antibodies used</td></tr><tr><td>Parasite Gas Mixture</td><td>By special order</td><td></td><td>3% CO&lt;sub&gt;2&lt;/sub&gt;, 1% O&lt;sub&gt;2&lt;/sub&gt;, balance N&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 &amp;#956;m filter unit</td><td></td><td></td><td></td></tr><tr><td>Glass slides</td><td></td><td></td><td></td></tr><tr><td>T25 flasks</td><td></td><td></td><td></td></tr><tr><td>T75 flasks</td><td></td><td></td><td></td></tr><tr><td>50 ml tubes</td><td></td><td></td><td></td></tr><tr><td>24 well culture plates</td><td></td><td></td><td></td></tr><tr><td>unplugged Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>plugged Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>sterile transfer pipettes</td><td></td><td></td><td></td></tr><tr><td>hemocytometer</td><td></td><td></td><td></td></tr><tr><td>flow cytometer (3 laser)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibodies used for flow cytometry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-TNF</td><td>eBiosciences</td><td>551487</td><td>FITC (fluorophore)
2 &amp;#956;l</td></tr><tr><td>Anti-IFN&amp;#947;</td><td>Biolegend</td><td>506507</td><td>PE (fluorophore) 
20 &amp;#956;l</td></tr><tr><td>Anti-CD8</td><td>Biolegend</td><td>300914</td><td>PE-Cy7 (fluorophore) 
1 &amp;#956;l</td></tr><tr><td>Anti-CD14</td><td>Biolegend; BD Biosciences</td><td>325624; 551487</td><td>Biotin (fluorophore) 
0.5 &amp;#956;l
Streptavin PE-TR (fluorophore) 
0.1 &amp;#956;l</td></tr><tr><td>Anti-CD56</td><td>Biolegend</td><td>318322</td><td>PerCP/Cy5.5 (fluorophore) 
5 &amp;#956;l</td></tr><tr><td>Anti-&amp;#947;&amp;#948;</td><td>Biolegend</td><td>331212</td><td>APC (fluorophore)
5 &amp;#956;l</td></tr><tr><td>Anti-CD3</td><td>Biolegend</td><td>300426</td><td>APC-Cy7 (fluorophore) 
5 &amp;#956;l</td></tr><tr><td>Anti-CD4</td><td>Biolegend</td><td>317424</td><td>Pacific Blue (fluorophore)
1 &amp;#956;l</td></tr></tbody></table>

an in vitro model for measuring immune responses to malaria in the context of hiv co-infection

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.

Grafikleri NKT hücreleri popülasyonu elde etmek CD56 +, CD3 + γδ- kapıları kullanan, NKT hücreleri IFNy üretimi (Şekil 2), seviyesini tasvir (veriler gösterilmemiştir). Hücreler boyamadan önce 72 saat süre ile kültüre edildi. Bir kez 100,000 CD3 + hücreleri sitometresi NK, NKT ve γδ hücrelerin (ilgi hücreleri) yeterince büyük popülasyonları elde etmek akışında elde edildi, boyandı. 5600 NKT hücrelerinin en az her bir grafik üzerinde gösterilir. TNF üretimi aynı şekilde elde edildi (veriler gösterilmemiştir). (-) PfRBC maruz kalanlarda grafikleri açık IFNy üretimi HIV (+), HIV ile karşılaştırıldığında bireylerden alınan hücrelerde daha düşük olduğunu göstermektedir. Sitometri analizi Flow çok subjektiftir. Her deney için tüm uygun kontroller olması esastır (bakınız Şekil 2). Arka plan düzeyi, sitokin boyama doğru sunulmasından sağlar FMO örnekleri, kullanılarak hesaplanır.PMA / İyonomisin ile uyarılan hücreler, bir pozitif kontrol olarak kullanılmıştır (veriler gösterilmemiştir). PMA ve İyonomisin T hücresi sitokin üretimin güçlü uyarıcısıdırlar. Bu örneklerde IFNy üretiminin eksikliği olur büyük olasılıkla boyama protokolü ile bir sorunu gösterir. Ancak, bu tür hücre canlılığı ya da inaktif reaktifler gibi diğer değişkenlerin de hatalı olabilir. <img alt="figür 1" src="/files/ftp_upload/52969/52969fig1.jpg" /> PBMC&#39;lerin konumunu gösteren Ficoll gradyan sonrası dönüş Şekil 1. tasviri. <img alt="Şekil 2," src="/files/ftp_upload/52969/52969fig2.jpg" /> Doğal Öldürücü T hücreleri tarafından Şekil 2. IFN γ üretimi. Akış şemaları CD56 + CD3 + γδ- hücreleri (5600 olaylarının az) üzerinde yolluk ile elde edilmiştir. (-) Ile uyarılan numunenin IFNy üretimi HIV tespit edilebilirP. falciparum kırmızı kan hücreleri enfekte. Bu sitokin yanıtı artık belirgin bir kronik HIV enfeksiyonu bağlamında olduğunu. <a href="https://www.jove.com/files/ftp_upload/52969/52969fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection at JoVE.com

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

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.

Bir<em&gt; İn Vitro</em&gt; HIV Co-enfeksiyon Bağlamında Sıtma için Bağışıklık Yanıtları Ölçüm Modeli

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

an-vitro-model-for-measuring-immune-responses-to-malaria-context-hiv

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

10.2K Görüntüleme.  University of Calgary. 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. 

Video: An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Bir<em&gt; In vitro</emOkumak&gt; Co-enfeksiyon Modeli<em&gt; Plasmodium falciparum</emİnsan İlköğretim Monosit kökenli Bağışıklık Hücrelerde&gt;-HIV-1 Etkileşimleri

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

İmmünoloji ve Enfeksiyon

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

Vitro Testi HIV-spesifik CD4 T hücre efektör işlevi üzerinde immünoregülatör yolları etkisini değerlendirmek için

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Doğal İletim üzerine Tüberküloz-Sıtma Koenfeksiyonu Çalışması için Deneysel Bir Model Mycobacterium tuberculosis Ve Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

Akış Cytometry tarafından Kemirgen Plasmodium parazitemi Vivo Değerlendirilmesi ve Merozoit Invasion

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

Insanlaşmış Fare Modelinde HIV enfeksiyonuna karşı kök hücre Tabanlı Engineered Bağışıklık

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Tıp

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Manyetik Rezonans Görüntüleme Kullanarak Deneysel Serebral Sıtma Bir Modelinde Ödem Gelişimi ve Mikrovasküler Patolojinin İn Vivo Takibi

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Akış SitometriSi-Bazlı Bir Test ile Plasmodium falciparum Parazitlerine Doğal Olarak Edinilmiş Fagositoz-Indükleyen Antikorların Ölçülmesi

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

In Vitro Sitotoksik Lenfositler Tarafından Plazmodyum ile Enfekte Kırmızı Kan Hücresi Öldürme Testi

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

Generation of an In Vitro Cell Culture Model of Malaria-HIV Co-Infection

Source: Finney, C. et al., <a href="https://app.jove.com/v/52969">An In Vitro&#160;Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates the generation of an in vitro malaria-HIV co-infection cell culture model. HIV-infected peripheral blood mononuclear cells co-cultured with Plasmodium falciparum parasitized erythrocytes potentially support each other&#39;s growth and provide insights into the immune responses and disease progression in HIV and malaria co-infections.

Sıtma-HIV Ko-Enfeksiyonunun İn Vitro Hücre Kültürü Modelinin Oluşturulması

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
CAUTION: Working with human blood ...

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