Este projeto foi financiado pela Case Western Reserve University fundos de inicialização fornecido para E. Jolly. Caramujos infectados foram fornecidos pelo Centro de Recursos esquistossomo, NIH-NIAID (Contrato n º HHSN272201000009I NIAID). Agradecemos também a Chris King e Ronald Blanton para o apoio na obtenção de caracóis infectados esquistossomo e para discussões úteis.

Medidas de segurança: Cercárias do S. mansoni pode penetrar diretamente na pele sem a necessidade de um corte ou poros, e causar a doença crônica da esquistossomose. S. mansoni é um nível de biossegurança 2 organismo, portanto, equipamento de proteção individual e medidas de segurança apropriadas devem ser seguidas quando se trabalha com cercaria infecciosa. A maioria dos equipamentos necessários para crescer e transformar schistosomes é padrão, com uma chave poucas exceções indicado na tabela ao final do protocolo. Todo o trabalho cercarial deve ser feito em uma sala isolada. Esquistossômulos devem ser cultivadas em um estéril capa de biossegurança, aprovado. Duas camadas de borracha nitrílica ou luvas de látex devem ser usadas, bem como uma bata de laboratório, um par de sapatos impermeáveis, calças sem furos ou rasgos, e mangas de protecção impermeável para seus pulsos e braços. Em caso de derrame acidental, pulverizar imediatamente a área contaminada com etanol 70% ou água sanitária a 10%, esfregando vigorosamente se em sua pessoa. Isso vai ser suficiente para desativar o parasita, mas todas as possíveis infecções devem ser relatados e levada a sério. Embora esquistossômulos não são inatamente infecciosa, diretrizes institucionais variam, e devem ser consultadas para determinar se BSL2 precauções são necessárias durante as fases da vida não-infecciosa. Desinfectar todas as superfícies que entram em contato com qualquer substância potencialmente infecciosos após o experimento é realizado e descarte de resíduos em um recipiente aprovado biohazard. NOTA: caixotes Cubra snail host 1 a 2 dias antes da queda e proteger da luz 1. Colheita cercárias a partir do host caracol <ol><li> Encher um copo limpo e livre de 500mL detergentes metade cheio com água destilada atingir a temperatura ambiente. Se trabalhar com caracóis hospedeiro infectado com miracídios em datas diferentes, use um copo separado para cada data de infecção (Isto é particularmente importante se os caracóis será reutilizado para futuros experimentos). O tamanho do copo também pode ser menor dependendo da experiência do usuário. </li><li> Coletar caramujos infectados hosts que você deseja galpão e depositá-los cuidadosamente no copo com um peixe de aquário padrão net e pinças de uso geral, conforme necessário. </li><li> Copo lugar em um derramamento de emergência bandeja de aproximadamente 8 a 12 polegadas sob uma fonte de luz incandescente. Deixe repousar por 1-2 horas sob a luz. Cercárias sair do caramujo hospedeiro quando expostos à luz brilhante. </li><li> Cuidadosamente filtrar caracóis a partir da mistura cercárias. Tomar precauções de segurança extra, como mistura é altamente infecciosa. Lembre-se, cercárias podem penetrar diretamente da pele humana! </li><li> Substituir mistura sob fonte de luz para cerca de 15 minutos, permitindo que qualquer questão dos resíduos de partículas para resolver. </li><li> Transferência de cercárias em um erlenmeyer limpo usando uma pipeta, evitando os destroços no fundo do copo. Colocar o balão em aproximadamente um ângulo de 45 ° no gelo no escuro por 45-60 minutos, permitindo cercárias para resolver em um canto. </li><li> Use uma pipeta de 50 mL para remover 75 a 90% do sobrenadante e depositá-lo no copo contendo lixívia 10% ou etanol 70%, tendo o cuidado de não deixar pingar gotas de água contendo cercárias da pipeta. </li><li> Agitar o erlenmeyer contendo cercárias suavemente para voltar a suspender as cercárias em solução. Use uma pipeta para transferir a amostra para 50 tubos estéreis ml. Coloque os tubos novamente no gelo por 20 minutos no escuro. Cercárias vai resolver para o fundo do tubo no escuro. </li></ol> 2. Transformação manual de cercárias em esquistossômulos Antes de começar: Faça RPMI 1640 completo (RPMI, 5% de soro fetal bovino, 1X Pen / Strep) e quente a 37 ° C. (É fundamental que o soro fetal bovino utilizado foi inativada pela incubação a 56 ° C durante 1 hora como schistosomes pode ser sensível para complementar). <ol><li> Em uma capa de risco biológico, remover o sobrenadante até 5 a 10 ml de solução em cada cônico de 50 ml utilizando uma pipeta ou outro dispositivo de vácuo. </li><li> Anexar um calibre 22 double-ended, luer lok emulsificante agulha para uma seringa de tamanho adequado. </li><li> Tirar lentamente a mistura para dentro da seringa. Coletar cercárias de todos os tubos de 50 ml em uma seringa. Esteja atento de gotejamento. </li><li> Cuidadosamente por sua vez seringa sobre como que o fim agulha não acoplada é para cima. Anexar outra seringa para este lado da agulha emulsificante. </li><li> Passe a mistura através da agulha de um total de 40 vezes (20 vezes ida e volta) para transformar o cercárias. </li><li> Posição das seringas na vertical de tal forma que a mistura está contida na seringa inferior e superior é a seringa vazia. Retire a seringa top e desinfectar em etanol 70%. </li><li> Gota a gota depósito mistura em um prato limpo alta 100 milímetros x 50 milímetros murada pirex ou prato de cristalização. Desinfectar agulha e seringa em Etanol 70%. </li><li> Adicionar 37 ° C RPMI 1640 completo de mídia de modo que ainferior da placa de Petri é completamente coberto, como se derramando um prato de caldo Luria. </li><li> Agite cuidadosamente para coletar esquistossômulos no centro da placa de Petri. Evitar respingos. Nota: Pode ser útil para desligar a luz do exaustor para esta etapa a mais adequada visualizar os esquistossômulos no centro. </li><li> Imediatamente depósito esquistossômulos concentrada em uma nova placa de cultura de células de 12 bem assim usando um conta-gotas estéril. </li><li> Repita 2.9 e 2.10 até esquistossômulos não estão mais presentes no centro do prato (cerca de 10 vezes). Depósito cada recolha subsequente em uma bem fresca. </li><li> Encher cada poço até metade (~ 1,5 mL) pré-aquecido com a mídia RPMI completo. </li><li> Visualize em um microscópio invertido para verificar a presença dos esquistossômulos e ausência de cercárias. </li><li> Crescer a esquistossômulos em uma incubadora de CO 2 set a 37 ° C e 5% CO 2. </li></ol> 3. Resultados representativos: Transferência pendente de esquistossômulos transformados em meios de cultura, visualização sob um microscópio de campo brilhante invertido (Ver tabela de equipamentos, por exemplo) deve render uma imagem semelhante à Figura 1, em que apenas esquistossômulos transformados estão presentes no poço. Contaminantes potenciais poderiam incluir cercárias intacta, caudas cortadas cercárias, ou outros detritos. Se nenhum microscópio invertido estiver disponível, um microscópio padrão ou mesmo um escopo dissecção será suficiente para visualizar os esquistossômulos. Note que este procedimento nem sempre transformar 100% de cercárias. <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="Figura 1"/> Figura 1: esquistossômulo Transformado <img src="/files/ftp_upload/3191/3191fig2.jpg" alt="Figura 2"/> Figura 2: cercárias e Tails Severed

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> N Engl J Med. 346, 1212-1220 (2002).</li><li>Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis+and+water+resources+development:+systematic+review,+meta-analysis,+and+estimates+of+people+at+risk.">Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk.</a> Lancet Infect Dis. 6, 411-425 (2006).</li><li>Savioli, L., Albonico, M., Engels, D., Montresor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis.">Progress in the prevention and control of schistosomiasis and soil-transmitted helminthiasis.</a> Parasitol Int. 53, 103-113 (2004).</li><li>King, C. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Schistosoma+mansoni+daughter+sporocysts+from+mother+sporocysts+maintained+in+synxenic+culture+with+Biomphalaria+glabrata+embryonic+(Bge)+cells.">Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells.</a> J Parasitol. 81, 714-722 (1995).</li><li>Colley, D. G., Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+simplified+method+for+the+production+of+schistosomules.">Schistosoma mansoni: simplified method for the production of schistosomules.</a> Exp Parasitol. 35, 44-51 (1974).</li><li>Lazdins, J. K., Stein, M. J., David, J. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+ultrastructure+of+early+transformation+of+skin-+and+shear-pressure-derived+schistosomules.">Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules.</a> Exp Parasitol. 51, 341-365 (1981).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. Chapter 19, 1-1 (2001).</li></ol>

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Vários problemas potenciais poderiam surgir durante o processo de transformação, no entanto, este protocolo é projetado para evitar a maioria destes problemas. Um risco potencial é o entupimento da agulha de emulsificante, que geralmente surge devido aos detritos que sobraram da colheita no passo 1.3. Certifique-se de permitir que todas as partículas para resolver no passo 1.5 e transferir a cercárias cuidadosamente no passo 1.6 para evitar este problema. Além disso, como descrito anteriormente, trabalhando dentro de um armário de segurança biológica, a utilização de equipamento de proteção individual (escudos, mesmo face) e evitar a pressão excessiva sobre as seringas é aconselhável. Moradia caudas cercarial pode ser visualizado na mídia, o que é indicativo da separação pobres durante o passo 2.9. As caudas apresentam um movimento espasmo-like e tem uma forma distinta (ver Figura 2), e desta forma são facilmente distinguíveis da esquistossômulos. As caudas podem ser removidos por lavagem a esquistossômulos em excesso de RPMI completo permitindo que os esquistossômulos para resolver. As caudas permanecerá flutuante e pode ser aspirado off. Se cercárias intactas estão presentes na mídia, a transformação no passo 2.5 não foi executado corretamente. Uma imagem de um cercaria intacta é retratado na Figura 3b, para referência. Se esse problema ocorre, assegurar que a agulha emulsionante a ser utilizado é do tamanho adequado. Além disso, o número de vezes que a mistura cercarial é passado através da agulha pode ser aumentado, apesar de 40 vezes é tipicamente muito mais do que suficiente para chegar perto dos 100% de corte das caudas cercárias. Transformação da cercária em esquistossômulos foi descrita pela primeira vez em 1974 por Colley e Wikel (13). Desde então, a preparação de esquistossômulos foi feito usando a estratégia do corte com uma agulha e seringa, conforme descrito neste protocolo, ou através de uma abordagem vórtex e separados por gradientes de Percoll (14, 15) ou rodando como demonstrado neste trabalho. Estas duas técnicas para preparar esquistossômulos são belamente descrito por Fred Lewis (16) e mais informações para cultura adicional de esquistossômulos de crescimento a longo prazo pode ser encontrado no NIAID esquistossomo Resource Center ( <a href="http://www.schisto-resource.org/">www.schisto-resource.org</a> ).

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> 22g x 2-7/8 &quot;agulha emulsionante </td><td> Fisher Scientific </td><td> 14-825-17G </td></tr><tr><td> RPMI 1640 1X Médio (500ml) </td><td> Invitrogen </td><td> 11835030 </td></tr><tr><td> De soro fetal bovino de calor (FBS) inativada </td><td> Invitrogen </td><td> 10082139 </td></tr><tr><td> Penicilina estreptomicina / (100X) </td><td> Invitrogen </td><td> 15070063 </td></tr><tr><td> Dish cristalizando (100mm) </td><td> Fisher Scientific </td><td> 08-741D </td></tr><tr><td> Objetivo geral Tweezers </td><td> Fisher Scientific </td><td> 10-316C </td></tr><tr><td> Petco Aquarium líquido artémia </td><td> Petco </td><td> SKU: 1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

Esquistossomo parasitas são os agentes causadores da esquistossomose, uma doença crônica e debilitante que afeta mais de 200 milhões de pessoas no mundo e ocupa o segundo lugar à malária entre as doenças parasitárias em termos de saúde pública e impacto sócio-econômico (1-4). Esquistossomo parasitas são vermes trematódeos com um ciclo de vida complexo intercâmbio entre uma vida parasitária em hospedeiros mamíferos e moluscos, com intervenção de natação livre etapas. Brevemente, de natação livre cercárias infectar um hospedeiro mamífero penetrando a pele com a ajuda de proteases secretadas, durante o qual o cercárias perdem a cauda, ​​transformando-se em esquistossômulos. Os esquistossômulos agora deve escapar do sistema imune do hospedeiro, desenvolver um intestino para a digestão das células vermelhas do sangue, e migrar embora os pulmões ea circulação portal rota para seu destino final no sistema porta hepática e, eventualmente, as veias mesentéricas (para S. mansoni) onde homens e mulheres par worms e mate, produzindo centenas de ovos por dia. Alguns dos ovos são excretados a partir do corpo em água doce, onde os ovos eclodem em nado livre miracídios (5-10). Os miracídios infectam espécies de caracóis específica e se transformar em mãe e filha esporocistos, que por sua vez, produzem cercárias infectantes, completando o ciclo de vida. Infelizmente, o ciclo de vida do S. mansoni não podem ser cultivadas in vitro, mas cercárias infectantes podem ser transformados em esquistossômulos, e os esquistossômulos podem ser cultivadas durante semanas para a análise do desenvolvimento do S. mansoni in vitro ou análise de microarray. Neste protocolo, nós fornecemos uma descrição visual de transformação cercárias e no cultivo in vitro de esquistossômulos. Nós eliminaram cercárias infecciosas da Biomphalaria glabrata caramujo hospedeiro e manualmente transformá-los em esquistossômulos, destacando suas caudas usando um emulsificante agulha dupla. A in vitro cercarial transformação e técnicas de cultura esquistossômulos descrito evitar o uso de uma série de mamíferos, o que simplifica a visualização de schistosomes e facilita a coleta do parasita para a análise experimental. Vitro na transformação e técnicas de cultivo de schistosomes ter sido feito há anos (11, 12), mas nenhum protocolo visuais têm sido desenvolvidos que estão disponíveis para toda a comunidade.

Watch this Scientific Journal Video about Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules at JoVE.com

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Nós descrevemos um<em&gt; In vitro</em&gt; Método para esquistossômulos cultura do parasita platelminto<em&gt; Schistosoma mansoni</em&gt;, Através da colheita e transformação de cercárias infectantes do caramujo de água doce Biomphalaria glabrata hospedeiro intermediário.

Transformação e Cercarial In vitro Cultivo de Schistosoma mansoni Esquistossômulos

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

cercarial-transformation-vitro-cultivation-schistosoma-mansoni

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JoVE Journal

Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

18.0K visualizações.  Case Western Reserve University. Nós descrevemos um In vitro Método para esquistossômulos cultura do parasita platelminto Schistosoma mansoni, Através da colheita e transformação de cercárias infectantes do caramujo de água doce Biomphalaria glabrata hospedeiro intermediário.

Vídeo: Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Usando Ovos de<em&gt; Schistosoma mansoni</em&gt; Como um<em&gt; Em vivo</em&gt; Modelo de Helmintos induzida por inflamação pulmonar

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Imunologia e Infecção

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

Isolamento em massa e cultivo In Vitro de Intramolluscan fases do sangue fascíola Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

Biologia do Desenvolvimento

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Transdução Transitória das Formas Estrofes estropidas de Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

Genética

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Utilização de<em&gt; Shigella flexneri</em&gt; Para estudar interações Autophagy-Cytoskeleton

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Isolamento e caracterização de vesículas extracelulares de adultos Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

Geração de Transgênicos e Nocautes em Espécies Strongyloides por Microinjeção

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Geração de Esporozoítos Geneticamente Modificados de Plasmodium berghei

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Isolamento e captação de células dendríticas de pequenas vesículas extracelulares de Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Nucleofection e In Vivo Propagação de Parasitas de Eimeria de Frango

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

Biologia

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Transformação e Cercarial In vitro Cultivo de Schistosoma mansoni Esquistossômulos

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