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.

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The authors have nothing to disclose.

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 (PfRBC) 17,18. Além disso, a activação de tipos de células, como as células NK, requer PfRBCs inteiros, e não funciona eficientemente com os lisados ​​de parasitas. Isto pode ser devido à necessidade de contacto directo entre o PfRBC e a de leucócitos ou pode ser devido à natureza instável dos ligandos derivados de parasitas que interagem com receptores de superfície celular 18. Culturas Malária PfRBC também deve ser bem sincronizados (Protocolo 4) antes da experiência. PfRBCs sincronizados melhorar a reprodutibilidade dos dados experimentais. Embora este protocolo desecretários uso de fase trofozóito PfRBC para co-cultura, isto pode facilmente ser modificado para o estudo de fase anel PfRBC. Leucócitos humanos podem ser infectados artificialmente com o HIV, e este método tem sido utilizado em estudos de investigação do HIV-malária co-infecção de 20. No entanto, isto não modelar a desregulação imune que resulta de infecção crónica pelo HIV. Neste sistema de co-cultura usamos CMSPs isolados a partir de participantes humanos que estão cronicamente infectados com HIV-1. Quando forem necessárias participantes para um estudo, é importante selecionar com cuidado essa população. Os critérios de inclusão e exclusão são vitais para garantir a variabilidade mínima e máxima reprodutibilidade. Os nossos critérios incluíram uma definição clara de infecção crônica pelo HIV (HIV-infectados por&gt; 1 ano, com CD4 + de células T declínio da contagem de&gt; 50 células / mm3 / ano) e à exclusão de qualquer pessoa com uma infecção concomitante. Isto assegurou que os resultados obtidos podem ser atribuídas assimlely para o efeito da infecção crónica pelo HIV, e não outra infecção. Além disso, uma vez que estavam interessados ​​em respostas imunitárias inatas à malária foram excluídos os doadores que tiveram infecção anterior malária. Controles não infectados pelo HIV também são utilizados para cada doador de HIV (+), em todos os pontos de tempo de amostragem, para permitir a normalização dos dados. Deve ser feita uma tentativa para combinar controles ao seu respectivo doador HIV (+) durante pelo menos idade, mas de preferência, também o sexo (embora em nosso estudo anterior 12 não observamos uma diferença significativa em subpopulações de células ou respostas de citocinas entre feminino e masculino HIV participantes não infectadas). Se a amostragem prospectivo está previsto manter o mesmo controle não infectados pelo HIV para cada ponto de tempo de amostragem é benéfico. As respostas podem variar de experiência para experiência devido a vários fatores, incluindo a saúde dos PfRBCs, o seu grau de sincronização, o nível de parasitemia e estágio de maturidade de PfRBCs, e nível de hematócrito. Cuidadodevem ser tomadas para manter como muitos destes consistente entre experimentos. Normalizando a um controlo não infectados pelo HIV permite alguma contabilidade para essas variáveis. Usando células frescas é fundamental para evitar resultados artificiais. Amostras de congelamento e descongelamento pode ter um impacto significativo na viabilidade das células 21,22, produção de citocinas e 23-26 fenotípica marcadores da superfície da célula 27. Se os monócitos são de particular interesse, é importante que o vidro não é utilizada ao longo do protocolo. Os monócitos aderir ao vidro e muitos outros materiais plásticos 28. Nós usamos pipetas de polipropileno de plástico, pipetas de transferência, e tubos em toda a minimizar a adesão de monócitos e remoção definitiva de nossas populações de células estudo. Ao configurar o ensaio de citometria de fluxo, qualquer combinação de anticorpos podem ser usados ​​dependendo das células de interesse. Estamos particularmente interessados ​​na produção de IFN e TNFa partir de células NK, as células NKT e as células T γδ. Este definido nosso painel de anticorpos. No entanto, se utilizando uma combinação diferente, é importante optimizar as quantidades de anticorpo para cada painel. Isso é vital para evitar dados errados. Além disso, para evitar a variabilidade e garantir a validade, FMOs são necessários para avaliar os níveis de citocinas fundo para cada condição (RBC, PfRBC, médio e PMA / ionomicina) e da população participante (HIV (-) e HIV (+), Figura 2). Medição da produção de citoquina intracelular in vitro geralmente resulta em elevados níveis de fundo, especialmente quando as células foram cultivadas antes da coloração. Uma vez que as condições de cultura de afectar os níveis de fundo, apropriados QOM assegurar o conhecimento do nível de fundo para cada amostra. Nosso suprimento de células foi limitado, portanto, nós projetamos nossos FMOs para conter todas as manchas superficiais, mas sem manchas intracelulares. Isto permite a comparação dos níveis de fundo específico de citocinas em todos os diferentes tipos de células estudados.Nós também correu uma única mancha por experimento para cada um dos marcadores de superfície para confirmar os seus níveis de fundo. O protocolo descrito é um versátil, que pode ser usado para examinar respostas dentro de horas ou dias, dependendo das células de interesse. Para a produção de citocina induzida por PfRBC óptima a partir de linfócitos inatos, nós medimos a citometria de fluxo de saída, após 2 ou 3 dias. Ao olhar para monócitos, pontos de tempo anteriores (1 ou 2 dias) são recomendados. Este sistema permite vários tipos de células e respostas celulares a ser distinguido por citometria de fluxo, as respostas secretoras de ser medidos em sobrenadantes celulares e perfis de expressão para ser avaliada em RNA extraído. Além disso, a utilização de anticorpos para bloquear os receptores específicos ou neutralizar as citocinas podem ser utilizadas neste sistema para dissecar ainda mais os mecanismos. Temos utilizado com sucesso IL-18 bloqueio do receptor para implicar IL-18 receptor de IFN-y nas respostas induzidas por PfRBC 12. Este sistema representa um realistaMétodo IC através do qual a avaliar uma série de respostas imunes inatas a malária no contexto da infecção pelo HIV.

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 monocï¿½icas também são prejudicados em HIV (+) doadores, mas recuperar pós-HIV terapia anti-retroviral.

<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.

Os gráficos apresentam os níveis de produção de IFN-y a partir de células NKT (Figura 2), usando CD56 + CD3 + γδ- portas para obtenção de uma população de células NKT (dados não mostrados). As células foram cultivadas durante 72 horas antes da coloração. Depois de coradas, 100.000 células CD3 + foram adquiridos no citómetro de fluxo para obter grandes populações suficientes de NK, células NKT e γδ (células de interesse). Um mínimo de 5.600 células NKT são exibidas em cada gráfico. A produção de TNF é obtido da mesma forma (dados não apresentados). Os gráficos demonstram claramente que a produção de IFNy em células é inferior a partir de HIV (+), em comparação com indivíduos HIV (-) indivíduos expostos a PfRBC. A citometria de fluxo análise é muito subjetivo. Por isso, é essencial ter todos os controles apropriados para cada experimento (veja a Figura 2). Os níveis de fundo são calculados com base nas amostras de FMO, o que permite uma verdadeira representação da coloração de citocinas.As células estimuladas com PMA / ionomicina foram utilizadas como um controlo positivo (dados não mostrados). PMA e ionomicina são fortes estimuladores da produção de citocina da célula T. Uma falta de produção de IFN-y nestas amostras seria mais provável indicar um problema com o protocolo de coloração. No entanto, outras variáveis, como a viabilidade celular ou reagentes inativos também podem estar em falta. <img alt="figura 1" src="/files/ftp_upload/52969/52969fig1.jpg" /> Figura 1. Representação de Ficoll pós gradiente rotação demonstrando a posição do PBMC. <img alt="Figura 2" src="/files/ftp_upload/52969/52969fig2.jpg" /> Figura 2. IFN γ produção por células assassinas T Natural. Os diagramas de fluxo foram obtidos por gating em CD56 + CD3 + γδ- células (mínimo de 5.600 eventos). Produção de IFN-y é detectável no HIV (-) de uma amostra com estimuladaP. falciparum glóbulos vermelhos infectados. Esta resposta de citocinas não aparente no contexto de uma infecção crônica do HIV é. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52969/52969fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</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.

Um<em&gt; In Vitro</em&gt; Modelo para medição de respostas imunes a Malária no Contexto do HIV co-infecção

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 visualizações.  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. 

Vídeo: 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.

Um<em&gt; In vitro</em&gt; Modelo de Co-infecção para Estudar<em&gt; Plasmodium falciparum</em&gt;-HIV-1 Interacções em monócitos humanos primários derivados de células imunes

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

Imunologia e Infecção

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.

In Vitro Ensaio para avaliar o impacto da imunorreguladora Pathways on específica para o HIV CD4 T efetoras celular Função

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.

Um modelo experimental para estudar a Tuberculose-Malária Coinfection sobre Transmissão Natural de Mycobacterium tuberculosis E 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.

Avaliando a inata Sensing de HIV-1 CD4 Infected<sup&gt; +</sup&gt; As células T por células dendríticas plasmocitóides Usando um<em&gt; Ex vivo</em&gt; Co-cultura do sistema.

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.

A avaliação in vivo de Rodent Plasmodium parasitemia e Merozoito Invasion por citometria de fluxo

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.

De células-tronco Baseado Imunidade Engineered contra infecção por HIV no modelo humanizado mouse

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

Medicina

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.

In Vivo Tracking of Edema Development and Microvascular Pathology em um Modelo de Malária Cerebral Experimental Usando Ressonância Magnética

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).

Medindo anticorpos indutores de fagocitose naturalmente adquiridos para parasitas plasmodium falciparum por um ensaio baseado em citometria de fluxo

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 Ensaio de Morte de Hemácias Infectadas por Plasmodium por Linfócitos Citotóxicos

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.remotexs.ntu.edu.sg/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.

Geração de um modelo de cultura de células in vitro de coinfecção malária-HIV

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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