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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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+changes+in+Schistosoma+mansoni+during+early+schistosomula+development+and+in+the+presence+of+erythrocytes.">Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes.</a> PLoS Negl Trop Dis. 4, (2011).</li><li>Basch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+Schistosoma+mansoni+in+vitro.+I.+Establishment+of+cultures+from+cercariae+and+development+until+pairing.">Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing.</a> J Parasitol. 67, 179-185 (1981).</li><li>Yoshino, T. P. &. a. m. p. ;. a. m. p., Laursen, J. 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. R., Sher, A. <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:+rapid+isolation+and+purification+of+schistosomula+of+different+developmental+stages+by+centrifugation+on+discontinuous+density+gradients+of+Percoll.">Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll.</a> Exp Parasitol. 53, 39-44 (1982).</li><li>Cousin, C. E., Stirewalt, M. A., Dorsey, C. 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>

Não há conflitos de interesse declarados.

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

Research

JoVE Journal

Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

17.9K 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

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

Em desencapsulamento vitro do HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imunologia e Infecção

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 ...

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 ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

Cultivo de<em&gt; Heligmosomoides polygyrus:</em&gt; Um imunomodulador Nematode Parasite e seus produtos secretados

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Transformação Biolistic de uma fluorescente Tagged Gene para o oportunista fúngica Pathogen<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Transformação de levedura probiótica e sua recuperação a partir gastrointestinais imunitário tecidos após a sonda oral em camundongos

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Os métodos in vitro para Comparando meta vinculativa e CDC indução Entre anticorpos terapêuticos: Aplicações em Análise Biosimilarity

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

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 ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Análise de interações entre endobióticos e microbiota intestinal humana usando sistemas de fermentação de banho in vitro

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Transfecção altamente eficiente de macrófagos primários com mRNA Transcrito in vitro

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...