Este estudo foi, em parte ou no todo, suporte Nacional Natural Science Foundation da China (31472187 e 31672550) e a ciência agrícola e programa de inovação de tecnologia da Academia Chinesa de Ciências agrárias.

Todos os experimentos com animais foram aprovados pelo Comitê de ética local do Instituto de investigação veterinária de Shanghai, Academia Chinesa de Ciências agrárias (número de licença: SHVRIAU-14-0101).
1. cultura in Vitro de Schistosomes
Nota: Schistosomes representam um risco biológico. Os trabalhadores devem usar luvas de látex vezes quando manipulação vermes, suspensões esquistossomóticos ou outros relacionados com materiais biológicos. Nova Zelândia coelhos foram infectados com cercárias ~ 2.000 via abdominal exposição. Schistosomes na fase hepática foram coletados de coelhos infectados com cercárias de S. japonicum a pós-infecção 28 dias por perfusão de fígado e veias mesentéricas. Os procedimentos de coleta de verme tem sido referenciados em publicações anteriores, relatórios de estudos em ratos18.
<ol><li>Recolha os parasitas em um copo de 500 mL. Lave-as e delicadamente 3 x 200 ml de pré-aquecido (37 ° C) PBS (pH 7,4).</li>
	<li>Separe pares de worm ~ 200 para cada prato de Petri de 90 mm. Então lave os parasitas e suavemente 2x com 50 mL de pré-aquecido (37 ° C) meio de cultura RPMI 1640 contendo 100 U de penicilina e 100 mg/mL de estreptomicina.</li>
	<li>Manter parasitas em 40 mL de meio de cultura RPMI-1640 pré-aquecido sem antibióticos a 37 ° C, abaixo dos 5% de CO2 em uma densidade de ~ 5 worm pares /mL para 2 h em 90 mm Petri dish. Notas: O meio RPMI 1640 não deve conter soro, caso contrário serão introduzida exógena vesículas extracelulares.</li>
</ol>2. previamente tratada condição Media
<ol><li>Recolher o meio de cultura e centrifugar o médio a 4 ° C, a 2.000 × g por 30 min remover os ovos e detritos tegmental de verme.</li>
	<li>Transferir o sobrenadante para um tubo de fresco e em seguida filtrar a solução através de um filtro de seringa 0,22 µm.</li>
	<li>Recolher a solução filtrada em um saco dializado (corte de peso molecular: 3.5kD) e em seguida Dialize a solução usando solução a 8% PEG8000 a 4 ° C durante a noite. Notas: Este passo é importante para enriquecer SjEVs para aumentar o rendimento de isolamento SjEVs.</li>
</ol>3. isolamento de SjEVs
<ol><li>Transferir a solução dializada para um novo tubo e adicione 0,5 volumes do reagente exossomo total isolamento.</li>
	<li>Misture a solução bem pela utilização do Vortex ou pipetagem para cima e para baixo para garantir a homogeneidade da solução.</li>
	<li>Incube a mistura entre 2 ° C a 8 ° C durante a noite.</li>
	<li>Centrifugue a mistura a 15.000 × g, durante 1 h, 2 ° c e 8 ° c.</li>
	<li>Aspirar e desprezar o sobrenadante. Notas: SVE constam o sedimento no fundo do tubo (não visível na maioria dos casos).</li>
	<li>Resuspenda o pellet EVs em 2 mL de PBS e em seguida armazenar a solução EVs a-80 ° C até uma análise mais aprofundada.</li>
</ol>4. caracterização de EVs pela mancha ocidental
<ol><li>Determine a concentração de proteína da amostra SjEVs, por exemplo, por meio de ensaio BCA.</li>
	<li>Prepare-se 10-20 µ g de EVs no buffer RIPA 22,5 µ l. Em seguida, adicione 7,5 µ l de 4x carregando buffer e calor durante 5 min à 95 ° C.</li>
	<li>Separe as proteínas SjEVs por gel de SDS-PAGE 12%.</li>
	<li>Transferência das proteínas à membrana PVDF e então sonda com anticorpos anti-CD63 (1:1, 000 diluição), seguidos por sondagem com IgG de coelho anti-HRP (1:5, 000 diluição).</li>
	<li>Utilizar o reagente de detecção de ECL para desenvolvimento de membrana e detectar os sinais por uma sistema de imagem de quimioluminescência.</li>
</ol>5. caracterização de EVs por microscopia eletrônica de transmissão
<ol><li>Adicionar 2 µ l de SjEVs isolado para 200 malha revestida formvar grades e depois deixe-o secar à temperatura ambiente.</li>
	<li>Mancha com 2% de ácido fosfotúngstico por 1 min e depois deixe-o secar à temperatura ambiente.</li>
	<li>Carregar as grades para porta-amostras de microscópio de elétron da transmissão e exposto a um feixe de elétrons 80 kV para captura de imagem.</li>
</ol>6. SjEVs rótulo com PKH67 e ensaio de absorção de células
Notas: Todas as etapas subsequentes foram realizadas à temperatura ambiente (20-25 ° C), salvo indicação em contrário.
<ol><li>Transferência de 5 µ g SjEVs solução para um tubo de polipropileno de fundo cónico e ajustar o volume de 12 mL pela adição de meio RPMI 1640.</li>
	<li>Centrifugue a SjEVs a 110.000 × g por 90 min a 4 ° C para SjEVs de Pelotas.</li>
	<li>Após a centrifugação, Aspire cuidadosamente o sobrenadante. Nota: A tintura de etanol PKH67 não deve ser adicionada diretamente para a pelota de SjEV, pois isso iria resultar em coloração heterogênea e viabilidade celular reduzida.</li>
	<li>Preparar uma solução a suspensão EV 2x, adicionando 1 mL de diluente C ao pellet SjEV; para garantir a completa dispersão, resuspenda com suave pipetagem.</li>
	<li>Antes de coloração em diluente C, prepare um fresco 2 x tintura solução adicionando 2 µ l da solução etanólica de tintura de PKH67 para 1 mL de diluente C num tubo de centrífuga de polipropileno e mistura bem para dispersar.</li>
	<li>Adicione rapidamente a 1 mL de 2x suspensão EV (etapa 6.4) a 1 mL de 2 x solução corante (passo 6.5) e imediatamente misture a amostra por pipetagem.</li>
	<li>Incube a mistura à temperatura ambiente durante 4 min.</li>
	<li>Parar a coloração adicionando 4 mL de FBS e então incubar durante 1 min.</li>
	<li>Adicione meio RPMI1640 para ajustar o volume de 12 mL e então centrifugar a 110.000 × g a 4 ° C por 90 min.</li>
	<li>Remover o sobrenadante e adicionar meio RPMI1640 para Ressuspender o PKH67 rotulado SjEV Pelotas.</li>
	<li>Centrifugar a 110.000 × g a 4 ° C por 90 min lavar uma vez e então adicione o PBS para Ressuspender as pelotas de SjEVs.</li>
</ol>7. ensaio de absorção de células SjEVs
<ol><li>Células de mamíferos de semente como NCTC clonagem células 1469 ou HEK293T em placas boas 6 (2 × 105 células por poço) e cultura das células durante a noite.</li>
	<li>Adicionar 5 µ g SjEVs PKH67-rotulados e cultura então células a 37 ° C, durante 2-4 h.</li>
	<li>Após a incubação, remover o meio e lavar as células com PBS duas vezes.</li>
	<li>Consertar as células com solução de formol 4% por 15 min e lavado duas vezes mais com PBS.</li>
	<li>Mancha do núcleo de células com 4, 6-diamidino-2-phenylindole (DAPI).</li>
	<li>Observe as células usando microscopia de fluorescência.</li>
</ol>

<ol>
	<li>Tetta, C., Ghigo, E., Silengo, L., Deregibus, M. C., Camussi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+an+emerging+mechanism+of+cell-to-cell+communication.">Extracellular vesicles as an emerging mechanism of cell-to-cell communication.</a> Endocrine. 44 (1), 11-19 (2013).</li><li>Abels, E. R., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+Extracellular+Vesicles:+Biogenesis,+RNA+Cargo+Selection,+Content,+Release,+and+Uptake.">Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake.</a> Cell Mol Neurobiol. 36 (3), 301-312 (2016).</li><li>Riaz, F., Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-like+vesicles+of+helminths:+implication+of+pathogenesis+and+vaccine+development.">Exosome-like vesicles of helminths: implication of pathogenesis and vaccine development.</a> Ann Transl Med. 5 (7), 175 (2017).</li><li>Zhang, H. G., Grizzle, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+cancer:+a+newly+described+pathway+of+immune+suppression.">Exosomes and cancer: a newly described pathway of immune suppression.</a> Clin Cancer Res. 17 (5), 959-964 (2011).</li><li>Colombo, M., Raposo, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annu Rev Cell Dev Biol. 30, 255-289 (2014).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNA+is+enriched+and+stable+in+exosomes:+a+promising+biomarker+for+cancer+diagnosis.">Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.</a> Cell Res. 25 (8), 981-984 (2015).</li><li>Marcilla, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+in+parasitic+diseases.">Extracellular vesicles in parasitic diseases.</a> J Extracell Vesicles. 3, 25040 (2014).</li><li>Cwiklinski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Extracellular+Vesicles+of+the+Helminth+Pathogen,+Fasciola+hepatica:+Biogenesis+Pathways+and+Cargo+Molecules+Involved+in+Parasite+Pathogenesis.">The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.</a> Mol Cell Proteomics. 14 (12), 3258-3273 (2015).</li><li>Buck, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+secreted+by+nematode+parasites+transfer+small+RNAs+to+mammalian+cells+and+modulate+innate+immunity.">Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity.</a> Nat Commun. 5, 5488 (2014).</li><li>Sotillo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+secreted+by+Schistosoma+mansoni+contain+protein+vaccine+candidates.">Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.</a> Int J Parasitol. 46 (1), 1-5 (2016).</li><li>Nowacki, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+and+small+non-coding+RNA-enriched+extracellular+vesicles+are+released+by+the+pathogenic+blood+fluke+Schistosoma+mansoni.">Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni.</a> J Extracell Vesicles. 4, 28665 (2015).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-like+vesicles+derived+by+Schistosoma+japonicum+adult+worms+mediates+M1+type+immune-+activity+of+macrophage.">Exosome-like vesicles derived by Schistosoma japonicum adult worms mediates M1 type immune- activity of macrophage.</a> Parasitol Res. 114 (5), 1865-1873 (2015).</li><li>Koh, Y. Q., Almughlliq, F. B., Vaswani, K., Peiris, H. N., Mitchell, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+enrichment+by+ultracentrifugation+and+size+exclusion+chromatography.">Exosome enrichment by ultracentrifugation and size exclusion chromatography.</a> Front Biosci (Landmark Ed). 23, 865-874 (2018).</li><li>Safdar, A., Saleem, A., Tarnopolsky, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+endurance+exercise-derived+exosomes+to+treat+metabolic+diseases.">The potential of endurance exercise-derived exosomes to treat metabolic diseases.</a> Nat Rev Endocrinol. 12 (9), 504-517 (2016).</li><li>Livshits, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+exosomes+by+differential+centrifugation:+Theoretical+analysis+of+a+commonly+used+protocol.">Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.</a> Sci Rep. 5, 17319 (2015).</li><li>Zhu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+extracellular+vesicles+containing+small+RNAs+from+the+eggs+of+Schistosoma+japonicum.">Release of extracellular vesicles containing small RNAs from the eggs of Schistosoma japonicum.</a> Parasit Vectors. 9 (1), 574 (2016).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+S.+japonicum+exosome-like+vesicles+reveals+their+regulatory+roles+in+parasite-host+interactions.">Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions.</a> Sci Rep. 6, 25885 (2016).</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. , 11 (2001).</li><li>Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+miRNAs:+roles+in+cancer+diagnosis,+prognosis+and+therapy.">Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy.</a> Adv Drug Deliv Rev. 81, 75-93 (2015).</li></ol>

Os autores não têm nada para divulgar.

Estudos recentes sobre EVs têm demonstrado que esquistossomíase que EVS desempenham um papel importante nas interações patógeno-hospedeiro3,9,12,de16. Para endereço ainda mais suas funções reguladoras, é essencial para isolar EVs de schistosomes. Aqui, descrevemos um método alternativo para SjEV isolamento. Este método produz um tamanho de ampla gama de SjEVs, de 100 nm a 400 nm, em ...

Vesículas extracelulares (EVs) são uma população de pequenas vesículas de membrana-limite encapsulado com várias proteínas, lípidos e ácidos nucleicos. Estudos recentes demonstraram que EVs desempenham um papel crucial na comunicação célula-célula e estão envolvidos na regulação de numerosos processos fisiológicos, incluindo desenvolvimento de célula, regulamento imune, angiogênese e da migração celular2, 3,4,5. Acumular evidências indica que EVs, circulando exosomes e sua carga de miRNA representa potenciais biomarcadores de determinadas doenças6.
Vários protozoários como Trichomonas vaginalis, Trypanosoma cruzie Leishmania spp., foram mostrados para ser capaz de secretar EVs; Além disso foram encontrados helmintos de secretar EVs em vivo hospeda7. EVs parasitárias foram mostrados para ser envolvido na manutenção da infecção, patogenicidade8e imune regra9. Recentes estudos em schistosomes ambos Schistosoma mansoni (S. mansoni) 10,11 e S. japonicum12 têm indicado que adultos schistosomes secretam exossomo como vesículas que podem ser envolvidos na regulamentação funcional de processos biológicos específicos.
Até à data, vários métodos têm sido utilizados para isolar vesículas extracelulares, tais como ultracentrifugação, ultrafiltração13, o uso de reagentes baseado em polímero, cromatografia de exclusão-tamanho e isolamento de immunoaffinity. Esses métodos diferentes possuem suas próprias vantagens e limitações de14. Geralmente, a ultracentrifugação é considerada o método padrão-ouro para isolamento de vesículas. No entanto, este método sofre a limitação do potencial de agregação de EV14.
Em schistosomes, foram relatados alguns métodos para isolamento de EV: Estes incluem ultracentrifugação11,12,10 e o uso de comerciais EV isolamento kit16 para vários estágios (ovos16, schistosomula11, schistosomes adulto10,12,17). Dado que aumentada é de uma vasta gama de tamanho de várias centenas de nanômetros a mil nanômetros, desenvolvemos um método alternativo que combina com o uso de centrífuga de bancada e um kit de isolamento comercial EV para isolar o SVE de ultrafiltração adultos de Schistosoma japonicum. O SVE isolado normalmente possuía um diâmetro de 100-400 nm e com êxito foram internalizado pelas células destinatários.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Total Exosome Isolation Reagent (from cell culture media)</td><td>ThermoFisher SCIENTIFIC</td><td>4478359</td><td>For isolating S. japonicum extracellular vesicles</td></tr><tr><td>PKH67</td><td>Sigma-aldrich</td><td>MINI67</td><td>For labeling Evs</td></tr><tr><td>RPMI 1640 Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11875119</td><td>For parasite culture</td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher SCIENTIFIC</td><td>15140122</td><td></td></tr><tr><td>0.22&amp;mu;m syringe filter</td><td>Merck</td><td>SLGP033RB</td><td></td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo SCIENTIFIC</td><td>68035</td><td></td></tr><tr><td>PEG8000</td><td>Sangon Biotech</td><td>A100159&amp;nbsp;</td><td></td></tr><tr><td>RIPA</td><td>Beyotime</td><td>P0013B</td><td></td></tr><tr><td>EMD Millipore&amp;trade; Immobilon&amp;trade; Western Chemiluminescent HRP Substrate (ECL)</td><td>Fisher scientific</td><td>WBKLS0100&amp;nbsp;</td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>4083</td><td></td></tr><tr><td>BCA kit</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>CD63 antibodies</td><td>Sangon Biotech</td><td>D160973-0025</td><td></td></tr><tr><td>PVDF</td><td>Bio-Rad</td><td>1704273</td><td></td></tr><tr><td>Formvar/Carbon 400 mesh</td><td>Ted Pella</td><td>01754-F</td><td></td></tr><tr><td>Phosphotungstic Acid</td><td>Ted Pella</td><td>19402</td><td></td></tr><tr><td>anti-mouse IgG-HRP</td><td>CWBIO</td><td>CW0102</td><td></td></tr><tr><td>NCTC clone 1469 cells</td><td>ATCC</td><td>ATCC&amp;reg; CCL-9.1&amp;trade;</td><td></td></tr><tr><td>FBS</td><td>HyClone</td><td>SV30087.02</td><td></td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE chemoluminescance imaging system</td><td>GE</td><td>ImageQuant LAS4000mini</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7600</td><td></td></tr><tr><td>Milli-Q water</td><td>Milli-Q</td><td>Milli-Q Elix</td><td></td></tr><tr><td>Eppendor Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5804R</td><td></td></tr><tr><td>Zetasizer Nano</td><td>Malvern</td><td>Zetasizer Nano ZS&amp;nbsp;</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima L-100 XP Ultracentrifuge</td><td></td></tr><tr><td>Incubator</td><td>ESCO</td><td>CCL-107B</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Zeiss Axin Observer Z1</td><td></td></tr></tbody></table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

Vesículas extracelulares (EVs) são vesículas membranosas lançadas por uma variedade de células no microambiente extracelular. SVE representa uma população de vesículas heterogêneas, cujo intervalo de tamanho entre 40 e 1.000 nm. Evidências acumuladas indicaram que EVs desempenham importantes funções reguladoras em interações patógeno-hospedeiro. Uma compreensão profunda da esquistossomíase EVs deve fornecer insights sobre os mecanismos subjacentes interações esquistossomíase-host, permitindo o desenvolvimento de novas estratégias contra a esquistossomose. Aqui, pretendemos estudar mais EVs funções em schistosomes apresentando um protocolo para o isolamento e caracterização de EVs de adultos Schistosoma japonicum (S. japonicum). SVE foram isolados em vitro o meio de cultura usando centrifugação combinada com um kit de isolamento exossomo comercial. Os isolados de S. japonicum EVs (SjEVs) normalmente possuem um diâmetro de 100-400 nm e são caracterizadas por microscopia eletrônica de transmissão e mancha ocidental. O uso de PKH67 SjEVs de tingir-etiquetados demonstrou que SjEVs são internalizados pelas células destinatários. No geral, o nosso protocolo fornece um método alternativo para isolar EVs de schistosomes adultos; o SjEVs isolado pode ser adequado para análise funcional.

Para quantificar o rendimento de SjEVs isolado usando o protocolo descrito, costumávamos ensaio da proteína BCA para acessar a concentração de proteína da SjEVs isolada de adultos schistosomes 28 dias. Conforme mostrado na tabela 1, a concentração de proteína SjEV variou de 208 µ g a 250 µ g por 100 mL de meio (tabela 1). O tamanho de partícula, conforme determinado pela análise de nanopartículas de Malvern, indicou que o isolado SjEVs variou...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolamento e caracterização de vesículas extracelulares de adultos Schistosoma japonicum

Aqui, apresentamos um protocolo para descrever o isolamento de vesículas extracelulares (EVs) em vitro culta médio 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 ...

isolation-characterization-extracellular-vesicles-from-adult

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

7.8K visualizações.  Ministry of Agriculture. Aqui, apresentamos um protocolo para descrever o isolamento de vesículas extracelulares (EVs) em vitro culta médio de adultos Schistosoma japonicum.

Vídeo: Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

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 ...

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 ...

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

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Um método de enriquecimento para pequenas vesículas extracelulares derivadas do tecido de câncer de fígado

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

Pesquisa do câncer

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Cromatografia de Exclusão de Tamanho para Separação de Vesículas Extracelulares de Meios de Cultura Celular Condicionada

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

Neurociência

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 ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purificação e Análise de Catadores Elegans Extracelulares

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Biologia

<meta charset="utf8"></head><body>Técnicas de refinamento para isolamento de exossomos derivados do baço murino

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

<meta charset="utf8"></head><body>Isolamento e caracterização de exossomos derivados de tecidos de baço de camundongo

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Isolamento e caracterização de exossomos derivados de tecidos de baço de camundongo

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

Isolamento e caracterização de vesículas extracelulares de adultos Schistosoma japonicum

Resumo

Explore mais vídeos

Capítulos neste vídeo

Transformação e Cercarial In vitro Cultivo de Schistosoma mansoni Esquistossômulos

Usando Ovos de

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

Um método de enriquecimento para pequenas vesículas extracelulares derivadas do tecido de câncer de fígado

Cromatografia de Exclusão de Tamanho para Separação de Vesículas Extracelulares de Meios de Cultura Celular Condicionada

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

Purificação e Análise de Catadores Elegans Extracelulares

Isolamento e caracterização de exossomos derivados de tecidos de baço de camundongo

Isolamento e caracterização de exossomos derivados de tecidos de baço de camundongo

Isolamento e caracterização de exossomos derivados de tecidos de baço de camundongo