Agradecemos a Francisco Candal, CDC Transferência de Tecnologia Escritório, Atlanta Geórgia para HBEC-5i células. Este trabalho foi financiado pelo Wellcome Trust (4 anos bolsa de estudo para doutorado e AC Fellowship sênior em ciência biomédica básica para JAR, conceder número 084226).

Recomendações gerais Cérebro humano microvascular cultura de células endoteliais (HBEC-5i) tem sido descrito anteriormente em 6, 8. P. parasitas falciparum foram cultivadas como no 9. Ambos HBEC-5i e P. culturas falciparum parasita devem ser mantidos em condições estéreis em todos os momentos. Todos os reagentes devem ser pré-aquecido a 37 ° C. Recomendamos que verifique regularmente se há contaminação por micoplasma 10 por PCR (Minevera Biolabs, as instruções do fabricante do seguinte). O protocolo está resumida na Figura 1. 1. Cultura de rotina da célula endotelial <ol><li> Prepare os reagentes necessários. </li></ol><table border="1"><tbody><tr><td> Médio para se preparar </td><td> Reagentes </td><td> Quantidade </td></tr><tr><td> &quot;DMEM incompleto&quot; </td><td> DMEM-F12 Ham </td><td> 500ml </td></tr><tr><td> &amp;nbsp; </td><td> L-glutamina 200mM </td><td> 5ml </td></tr><tr><td></td><td> Penicilina estreptomicina / 100X </td><td> 5 ml </td></tr><tr><td></td><td> NaOH 1M </td><td> 1,3 ml (ajustar o pH a 7.4) </td></tr><tr><td> &quot;DMEM completo&quot; </td><td> &quot;DMEM incompleto&quot; </td><td> 450ml </td></tr><tr><td></td><td> Fetal bovino soro inativado pelo calor </td><td> 50ml </td></tr><tr><td></td><td> Suplemento de crescimento da célula endotelial </td><td> 5 ml </td></tr></tbody></table> <ol start="2"><li> Cultura HBEC-5i num frasco de 25 centímetros 2 perfurada com 10ml meio DMEM completo em um 37 ° C incubadora com 5% de CO 2. </li><li> Células passagem quando eles se tornam confluentes. Remover o meio de idade por sucção e lavar duas vezes com DMEM incompleta ou cultura de tecidos grau PBS (Ca 2 + e Mg 2 + livre) pré-aquecido a 37 ° C. </li><li> Adicionar 1 ml de pré-aquecido Tentepsin-EDTA (0,025% Tripsina, 0,5 mM EDTA), agite para cobrir a totalidade do frasco e incubar por ~ 2 min a 37 ° C. </li><li> Seleção em microscópio invertido que pelo menos 90% das células foram destacados. Se necessário, delicadamente bater o fundo do frasco para retirar células aderentes. Adicionar 10 ml de meio completo DMEM para impedir as células de tripsina e transferir para um tubo cônico 15ml. Centrifugar por 4 min a 300 g à temperatura ambiente (RT). </li><li> Desprezar o sobrenadante e ressuspender o pellet com 10 ml de meio completo DMEM. Pipeta a solução cima e para baixo completamente ressuspender as células. </li><li> Adicionar 1 ou 2 ml da suspensão de células para um balão nova cultura e adicione 8ml de meio fresco para manter a cultura. Avaliar o crescimento da cultura todos os dias sob um microscópio invertido e mudança de médio a cada 2 ou 3 dias antes que ele fica amarelo. </li></ol> 2. Preparar células endoteliais para uma seleção <ol><li> Dois dias antes do dia da seleção e<html> n, adicione fibronectina diluído em PBS estéril (2 mg / cm 2) em um (ou mais) 60 mm placa de Petri. Incubar o prato por 5 a 20 min a 37 ° C, em seguida, remova a solução fibronectina, que pode ser armazenado a 4 ° C por um mês e re-utilizado uma vez. </li><li> Células passagem como descrito nas seções 1.3. para 1,5. Assumindo 100% de células confluentes foram destacados e ressuspenso em 10ml meio DMEM completo (seção 1.6., Ressuspender com um volume equivalente de médio se a confluência é menor, por exemplo, ressuspender com 8ml se a confluência foi de 80%), adicionar 1,5 ml da suspensão células à placa de Petri revestidas fibronectina e outro de 1,5 ml de meio completo DMEM. </li><li> Coloque a placa de Petri semeadas na incubadora. Nota: Idealmente, a confluência será em torno de 90% no momento da seleção dois dias depois. </li><li> Opcional. Para ativar HBEC-5i, adicione o TNF de citocina (fator de necrose tumoral) em uma concentração final de 50μg/ml 24 horas antes da seleção. </li></ol>e_title &quot;&gt; 3. Plasmodium falciparum cultura de rotina <ol><li> Prepare os reagentes necessários (veja a tabela aqui abaixo). Prepare células vermelhas do sangue (RBC), separando sangue total (grupo O +) pela passagem através de um filtro de remoção de leucócitos (ver &quot;Métodos de Pesquisa em Malária&quot; publicação 11 para cultura geral parasitas da malária). Lavar RBC duas vezes por centrifugação a 400 g por 5 min e ressuspensão com 10 ml RPMI incompleta. Mantenha RBC lavado a 4 ° C em meio incompleta em hematócrito de 50%. </li></ol><table border="1"><tbody><tr><td> Médio para se preparar </td><td> Reagentes </td><td> Quantidade </td></tr><tr><td> &quot;RPMI incompleto&quot; </td><td> RPMI 1640 (com bicarbonato) </td><td> 500ml </td></tr><tr><td></td><td> Hepes 1M </td><td> 12.5ml </td></tr><tr><td></td><td> Glicose a 20% </td><td> 5ml </td></tr><tr><td></td&gt; <td> L-glutamina 200mM </td><td> 5ml </td></tr><tr><td></td><td> Gentamicina 50mg/ml </td><td> 250μl </td></tr><tr><td></td><td> NaOH 1M </td><td> 0,7 ml (ajustar o pH a 7.2) </td></tr><tr><td> &quot;RPMI completo&quot; </td><td> &quot;RPMI incompleto&quot; </td><td> 450ml </td></tr><tr><td></td><td> Agrupados humano (não imunes) de soro </td><td> 50ml </td></tr></tbody></table> <ol start="2"><li> Cultura P. falciparum infectados RBC com meio completo RPMI a 37 ° C com 3% de CO 2, 1% O 2, e 96% N 2. hematócrito em 2% e incubar Faça Giemsa esfregaço 11 diárias para avaliar o estágio de desenvolvimento de parasitas (Figura 2). </li><li> Regularmente (aproximadamente uma vez por semana) sincronizar cultura por tratamento sorbitol 12. O dia antes do ensaio da selecção, objectivo para uma cultura anel de estágio na parasitemia 5% ou mais (de preferência pelo menos 10%). </li></ol> 4. Seleção de P. falciparum para cytoadhesion às células endoteliais <ol><li> No dia do ensaio, a cultura deve ser parasita na fase trofozoíto pigmentados (Figura 2) (idealmente parasitemia 10%), enquanto HBEC-5i cultura deve ser de 50 a 100% confluência (idealmente 90%). Volume celular 30μl repleta de cultura parasita é necessário por HBEC-5i placa de Petri. </li><li> Lavar parasitas duas vezes por centrifugação (500 g por 5 min) 1,5 ml da cultura parasita. Elimine o sobrenadante e ressuspender com 10ml de preparados na hora, aquecido meio DMEM, incompleta. Repita a lavagem pela segunda vez. Ressuspender o volume celular 30μl embalado com 1,5 ml de DMEM incompleta com BSA 1%. </li><li> Lave o HBEC-5i placa de Petri revestidas por duas vezes aspiração médio e adicionando 3 ml de DMEM incompleta. </li><li> Adicionar a solução de parasitas para o prato HBEC-5i e incubar a 37 ° C por 75min. Ressuspender parasitas duas vezes (aos 30 e 60 min) durante a incubaçãosuavemente balanço o prato nas quatro direções, bem como no sentido horário e anti-horário. </li><li> Após a incubação, lavar o prato 5 vezes por meio de aspiração, usando um plástico Pasteur pipeta para adicionar 3 ml de meio DMEM quente incompleta, e de balanço suave. Se muitos pratos estão sendo utilizados, mantê-los em uma superfície quente, como um grande frasco cheio de água a 37 ° C. </li><li> Verifique o prato sob um microscópio invertido. Se muitos RBC não infectados ainda são visíveis, não lava mais como descrito acima. Lidar com a placa de Petri com muito cuidado para evitar qualquer risco de contaminação. </li><li> Remova o meio do prato e adicionar 3 ml de meio RPMI quente completo com volume celular 40μl repleta de uRBC fresco. </li><li> Coloque o prato em uma câmara hermética de incubação, o gás por 3 min e colocar a câmara em uma incubadora a 37 ° C durante a noite. </li><li> No dia seguinte, colher os parasitas por lavagem com RPMI médio incompletos, de um modo semelhante ao descrito na seção 4.4. mas de forma mais vigorosa. Keep todos usados ​​médio (contendo ressuspenso RBC) em um tubo de 15ml cônico. Seleção em microscópio invertido que todos RBC foram removidos do prato. </li><li> Pellet os parasitas, desprezar o sobrenadante, ressuspender em meio RPMI 5ml completo e coloque a mistura em um frasco de cultura normal. </li></ol> 5. Resultados representante Parasitas não selecionados mostram um baixo nível de ligação a HBEC-5i (Figura 3A). Assim, após a primeira rodada de seleção, os parasitas poucos serão colhidos e pode demorar até um mês de cultura para chegar a uma parasitemia suficiente para a segunda rodada de seleção. Após cada rodada de seleção, mais parasitas e mais se ligam a células endoteliais e as seleções pode ser repetido em intervalos de tempo mais curto. Após 4-5 rodadas de seleção, as populações de alta ligação parasita são obtidos (Figura 3). Em nossas mãos, a ligação de HB3-HBEC para HBEC-5i ou ativado HBEC TNF-5i era de simhomólogos de nível (dados não mostrados). O protocolo descrito aqui foi testado com 4 p. cepas falciparum: HB3, IT/FCR3, 3d7 e DD2 (Tabela 1). Apenas o último não provou ser capaz de se ligar a HBEC-5i, mesmo depois de cinco rodadas de seleção. HB3 parasitas também foram selecionados para a citoaderência Humanos dérmica e pulmonar células endoteliais microvasculares (HDMEC e HPMEC). Após 4 rodadas de seleção, usando o método descrito aqui, uma população de alto-binding foi obtido em ambos os HDMEC e HPMEC (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/3122/3122fig1.jpg" /> Figura 1. Visão geral do processo de seleção. <img alt="Figura 2" src="/files/ftp_upload/3122/3122fig2.jpg" /> Estágios figura 2. Desenvolvimento de P. falciparum glóbulos vermelhos infectados. Esfregaço Giemsa visualizados sob microscópio com ampliação de 1000X. <img alt="Figura 3" src="/files/ftp_upload/3122/3122fig3.jpg" /> Figura 3. Exemplo típico de parasitas ligação a HBEC-5i. (A) A camada azul é HBEC-5i fixado em glutaraldeído e corados com Giemsa, visualizados sob microscópio com ampliação de 1000X. Fotos foram tiradas após uRBC e iRBC unbound foram lavados. O painel esquerdo mostra um único P. falciparum HB3 (não selecionados) parasita obrigado a HBEC-5i. No painel direito os parasitas HB3-HBEC tinha sido seleccionado para cinco rounds. (B) Os dados representam a média de dois experimentos independentes, cada uma realizada em duplicata. O número de parasitas obrigados por célula endotelial foi contado. <img alt="Tabela 1" src="/files/ftp_upload/3122/3122table1.jpg" /> Tabela 1. Resumo das cepas de Plasmodium falciparum que foram selecionados com sucesso para a ligação com HBEC Nota-5i. HB3 que também foi selecionado no TNF-activated HBCE-5i. Após 5 rodadas de seleção, a tensão DD2 não mostrou aumento na ligação para HBEC-5i comparação com unselected-DD2. <img alt="Figura 4" src="/files/ftp_upload/3122/3122fig4.jpg" /> Figura 4. Ligação do P. falciparum HB3 parasitas para dérmica (HDMEC) e pulmonar (HPMEC) células endoteliais, antes e depois de quatro rodadas de seleção. Embora o meio de cultura para HDMEC e HPMEC ligeiramente diferente (veja as instruções do fornecedor), o protocolo usado para a seleção foi idêntica como com HBEC-5i. Fotos tiradas com ampliação de 400X. <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> DMEM-F12 Ham </td><td> Sigma </td><td> D6421 </td><td> Por meio completo DMEM </td></tr><tr><td> L-glutamina 200mM </td><td>GIBCO </td><td> 25030 </td><td> Para DMEM e meio completo RPMI </td></tr><tr><td> Penicilina estreptomicina / 100X (10.000 unidades / ml e 10mg/ml) </td><td> ScienCell </td><td> 0503 </td><td> Por meio completo DMEM </td></tr><tr><td> Fetal bovino soro inativado pelo calor </td><td> ScienCell </td><td> 0025 </td><td> Por meio completo DMEM </td></tr><tr><td> Tripsina-EDTA (0,025% Tripsina, 0,5 mM EDTA) </td><td> ScienCell </td><td> 0103 </td><td></td></tr><tr><td> Suplemento de crescimento da célula endotelial </td><td> ScienCell </td><td> 1052 </td><td> Por meio completo DMEM </td></tr><tr><td> Cultura de tecidos tratados 60 milímetros X 15 milímetros placas de Petri </td><td> BD </td><td> 353002 </td><td></td></tr><tr><td> A fibronectina humana </td><td> Millipore </td><td> FC010 </td><td> Use a 2 mg / cm 2 </td></tr><tr><td> TNF </td><td> R &amp;D Systems </td><td> 210-TA </td><td> opcional, use a 50 mg / ml </td></tr><tr><td> RPMI 1640 </td><td> Lonza </td><td> BE12-167f </td><td> Por meio completo RPMI </td></tr><tr><td> Gentamicina 50mg/ml </td><td> Lonza </td><td> 17-518Z </td><td> Por meio completo RPMI </td></tr><tr><td> Hepes 1M </td><td> Lonza </td><td> BE17-737E </td><td> Por meio completo RPMI </td></tr><tr><td> HDMEC </td><td> ScienCell </td><td> 2000 </td><td> Linhagem de células primárias </td></tr><tr><td> HPMEC </td><td> ScienCell </td><td> 3000 </td><td> Linhagem de células primárias </td></tr><tr><td> HBEC-5i </td><td></td><td></td><td> Obtido a partir de Francisco Candal (fcandal@cdc.gov) </td></tr></tbody></table> Tabela 2. Materiais

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Não temos nada a revelar.

A marca da malária cerebral é o seqüestro de P. falciparum iRBC dentro do cérebro microvasculatura 2, 3. No entanto, em culturas in vitro de P. falciparum apenas mal cytoadhere para HBEC-5i, um modelo de cérebro humano endotélio microvascular. Aqui desenvolvemos um ensaio simples para enriquecer uma população para a ligação com HBEC-5i, um mais &quot;in-vivo, como&quot; fenótipo. Três em cada 4 p. cepas falciparum foram selecionados com sucesso usando este método. Além disso, HB3 também foi selecionado em HDMEC e HPMEC, indicando que este protocolo pode ser usado para vários tipos e parasita das células endoteliais. Diferentes hipóteses podem explicar a falta de ligação com a cepa DD2. O mais provável é o fato de que esta linha parasita é knobless, que impede a profundamente citoaderência 13, 14. Recomendamos parasitas cultura não selecionados ao longo do pro seleçãocesso para fornecer um controle para comparação. Isto irá permitir, por exemplo, comparando o transcriptoma de parasitas vinculativa e não vinculativa, com a esperança de descobrir candidatos ligante do parasita. TNF é uma citocina encontrados em nível elevado em pacientes com malária cerebral e foi mostrar para induzir a expressão de proteínas de superfície muitas das HBEC-5i (Claessens et al, em preparação e 8, 15, 16). Neste caso, a quantidade de iRBC ligada foi semelhante em HBEC-5i comparação com ativado HBEC-5i. Este &quot;modelo de sequestro&quot; também pode ser usado para estudar a interação molecular entre os iRBC e as células endoteliais, bem como o efeito do suposto anti-citoaderência drogas. Neste caso, recomendamos plating HBEC-5i em poços menores, tais como &quot;slides oito bem-câmara&quot; (BD 354628) ou &quot;CultureWell&quot; (Sigma C7735-20EA).

selection of plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells

A maioria das mortes por malária humana é causada por parasitas do sangue estágio Plasmodium falciparum. Malária cerebral, a complicação mais risco de vida da doença, é caracterizada por um acúmulo de Plasmodium falciparum glóbulos vermelhos infectados (iRBC) na fase trofozoíto pigmentada na microvasculatura do cérebro 2-4. Esta obstrução microvascular (seqüestro) leva à hipóxia, acidose e prejudiciais citocinas inflamatórias (revisto em 5). Seqüestro também é encontrado na maioria dos tecidos microvascular do corpo humano 2, 3. O mecanismo pelo qual iRBC anexar às paredes dos vasos sanguíneos é ainda mal compreendida. O imortalizado Human Brain microvascular linhagem de células endoteliais (HBEC-5i) tem sido usado como um modelo in vitro da barreira hemato-encefálica 6. No entanto, o Plasmodium falciparum iRBC anexar apenas mal ao HBEC-5i in vitro, ao contrário do sequestrat densaion que ocorre em casos de malária cerebral. Por isso, desenvolveu um ensaio panning para selecionar (enriquecer) p. vários cepas falciparum para adesão ao HBEC-5i, a fim de obter populações de alto-binding parasitas, mais representativa do que ocorre in vivo. Uma amostra de uma cultura parasita (mistura de RBC iRBC e não infectadas) na fase de trofozoíto pigmentada é lavado e incubado em uma camada de HBEC-5i cultivados em uma placa de Petri. Após a incubação, o prato é cuidadosamente lavado e livre de uRBC iRBC desacoplado. URBC fresco são adicionadas ao iRBC alguns anexados a HBEC-5i e incubadas durante a noite. Como parasitas estágio esquizonte estourar, merozoítos reinvade RBC e estes parasitas estágio anel são colhidas no dia seguinte. Parasitas são cultivadas até material suficiente é obtida (geralmente 2 a 4 semanas) e uma nova rodada de seleção pode ser realizada. Dependendo do P. falciparum tensão, 4-7 rodadas de seleção são necessários a fim de obter uma populaçãoonde a maioria dos parasitas se ligam a HBEC-5i. O fenótipo de ligação é progressivamente perdida depois de algumas semanas, indicando uma mudança na expressão do gene variante antígeno de superfície, assim, a seleção regulares sobre HBEC-5i é necessária para manter o fenótipo. Em resumo, nós desenvolvemos um ensaio de renderização seleção P. parasitas falciparum uma forma mais &quot;cerebral da malária adesivo&quot; fenótipo. Fomos capazes de selecionar 3 de 4 p. cepas falciparum em HBEC-5i. Este ensaio também tem sido usados ​​com sucesso para selecionar os parasitas para a ligação com humanos dérmica e pulmonar de células endoteliais. Importante, este método pode ser usado para selecionar populações de tecidos específicos do parasita, a fim de identificar ligantes do parasita candidato para a ligação ao endotélio cerebral. Além disso, este ensaio pode ser utilizado para triagem de suposto seqüestro de anti-drogas 7.

Watch this Scientific Journal Video about Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells at JoVE.com

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Um<em&gt; In vitro</em&gt; Modelo para o seqüestro de malária cerebral é descrita<sup&gt; 1</sup&gt;.<em&gt; Plasmodium falciparum</em&gt; Glóbulos vermelhos infectados são selecionados para a ligação com células cerebrais humanas imortalizadas microvascular endoteliais. Os parasitas selecionados mostram um fenótipo distinto. O processo de seleção pode ser aplicado usando diversos<em&gt; P. falciparum</em&gt; Cepas e linhagens de células endoteliais.

Seleção de Plasmodium falciparum Parasitas para Cytoadhesion para células do cérebro humano endotelial

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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Research

JoVE Journal

Immunology and Infection

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

18.9K visualizações.  University of Edinburgh. Um In vitro Modelo para o seqüestro de malária cerebral é descrita 1. Plasmodium falciparum Glóbulos vermelhos infectados são selecionados para a ligação com células cerebrais humanas imortalizadas microvascular endoteliais. Os parasitas selecionados mostram um fenótipo distinto. O processo de seleção pode ser aplicado usando diversos P. falciparum Cepas e linhagens de células endoteliais.

Vídeo: Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

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

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Um protocolo simples para agregação plaquetária mediada por<em&gt; Plasmodium falciparum</emEritrócitos&gt; infectados em uma configuração com poucos recursos

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Isolamento e Análise de Cérebro-sequestrados de Leucócitos<em&gt; Plasmodium berghei</em&gt; ANKA infectadas Mice

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Separação de<em&gt; Plasmodium falciparum</em&gt; Fase Tardia eritrócitos infectados por meio magnético

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitativo<em&gt; In vitro</em&gt; Ensaio medir a adesão de neutrófilos ativados primárias células humanas endoteliais microvasculares em condições estáticas

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

High Yield purificação<em&gt; Plasmodium falciparum</em&gt; Merozoítos Para uso em opsonizantes Anticorpo Ensaios

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

Métodos para investigar o papel de Regulamentação dos Pequenos RNAs e Ribosomal Ocupação de<em&gt; Plasmodium falciparum</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Infecção pneumocócica de células endotélias humanas primárias em fluxo constante

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Neisseria meningitidis Infecção de Células-Tronco Pluripotentes Induzidas Derivadas do Endotélio Cerebral

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...