Questo progetto è stato finanziato dalla Case Western Reserve fondi di avvio Università fornite E. Jolly. Lumache infette sono stati forniti dal Centro Risorse schistosoma, NIAID-NIH (NIAID Contratto N. HHSN272201000009I). Ringraziamo anche Chris King e Ronald Blanton per il supporto nel garantire infetti lumache schistosoma e per utili discussioni.

Misure di sicurezza: Cercarie schistosoma può direttamente penetrare la pelle senza la necessità di un taglio o pori, e causare la schistosomiasi cronica. S. mansoni è un livello di biosicurezza 2 organismo, quindi un equipaggiamento di protezione individuale e le misure di sicurezza adeguate devono essere seguite quando si lavora con Cercaire infettive. La maggior parte delle attrezzature necessarie per crescere e trasformare schistosomi è standard, con poche eccezioni chiave indicato nella tabella alla fine del protocollo. Tutti i lavori cercarie dovrebbe essere fatto in una stanza isolata. Schistosomules dovrebbe essere colta in una sterile, cappuccio approvato biosicurezza. Due strati di nitrile o guanti di lattice dovrebbero essere indossati, così come un camice, un paio di scarpe impermeabili, pantaloni senza buchi o strappi, e protettori manica impermeabile all&#39;acqua per i polsi e le braccia. In caso di sversamento accidentale, spruzzare immediatamente l&#39;area contaminata con etanolo al 70% o 10% di candeggina, strofinando vigorosamente se sulla vostra persona. Questo sarà sufficiente per disabilitare il parassita, ma ogni eventuale infezione devono essere segnalati e presi sul serio. Anche se non sono congenitamente schistosomules infettive, le linee guida istituzionali variano, e dovrebbe essere consultato per determinare se BSL2 precauzioni sono necessarie durante le fasi della vita non infettive. Disinfettare tutte le superfici che vengono a contatto con sostanze potenzialmente infettivi dopo l&#39;esperimento viene eseguito e gettare rifiuti in un contenitore approvato rischio biologico. NOTA: Copertina cassonetti ospitare lumaca 1 a 2 giorni prima del distacco e proteggere dalla luce 1. La raccolta cercarie dall&#39;host lumaca <ol><li> Riempire un ambiente pulito, coppa 500mL circolazione dei detergenti a metà pieno di acqua distillata equilibrati a temperatura ambiente. Se si lavora con le lumache host infetto con miracidi in date diverse, utilizzare un bicchiere separato per ogni data di infezione (Questo è particolarmente importante se le lumache saranno riutilizzati per futuri esperimenti). La dimensione del bicchiere può anche essere più piccolo a seconda della esperienza dell&#39;utente. </li><li> Raccogliere gli host infetti lumaca che si desidera versare e accuratamente depositarli nel bicchiere con un pesce d&#39;acquario standard di rete e pinze uso generale, se necessario. </li><li> Bicchiere posto su un vassoio d&#39;emergenza versare circa 8 a 12 pollici sotto una lampada a incandescenza. Lasciate riposare per 1-2 ore sotto la luce. Cercarie uscire l&#39;host lumaca quando esposto alla luce. </li><li> Con attenzione filtrare lumache dalla miscela cercarie. Prendere misure di sicurezza supplementari, come miscela altamente contagiosa. Ricorda, cercarie possono direttamente penetrare la pelle umana! </li><li> Sostituire mischia fonte di luce per circa 15 minuti, consentendo ad ogni questione rifiuti particelle di stabilirsi. </li><li> Trasferimento cercarie in un beuta pulita utilizzando una pipetta, evitando i detriti sul fondo del bicchiere. Porre il pallone a circa 45 ° su ghiaccio al buio per 45 - 60 minuti, permettendo cercarie di stabilirsi in un angolo. </li><li> Utilizzare una pipetta 50 ml di togliere 75 al 90% del supernatante e depositarlo in un bicchiere contenente candeggina al 10% o 70% di etanolo, facendo attenzione a non lasciare che le gocce d&#39;acqua che contiene a goccia cercarie dalla pipetta. </li><li> Agitare la beuta contenente cercarie delicatamente per risospendere le cercarie in soluzione. Utilizzare una pipetta per trasferire il campione al sterile 50 ml provette coniche. Posizionare i tubi di nuovo sul ghiaccio per 20 minuti al buio. Cercarie si depositerà verso il fondo della provetta al buio. </li></ol> 2. Trasformazione manuale di cercarie di schistosomules Prima di iniziare: Fare RPMI 1640 completo (RPMI, 5% siero fetale bovino, 1X Pen / Strep) e calda a 37 ° C. (E &#39;fondamentale che il siero fetale bovino utilizzato è stato inattivato tramite incubazione a 56 ° C per 1 ora come schistosomi possono essere sensibili a complemento). <ol><li> In una cappa di rischio biologico, rimuovere il surnatante fino a 5 a 10 ml di soluzione in ogni 50 ml con una pipetta o un dispositivo vuoto. </li><li> Collegare un AWG 22 double-ended, luer lok emulsionare l&#39;ago di una siringa di dimensioni appropriate. </li><li> Lentamente disegnare impasto nella siringa. Raccogliere cercarie da tutte le provette da 50 ml in una siringa. Essere attenti di gocciolamento. </li><li> Con attenzione volta siringa sopra tale che l&#39;estremità ago separato è in alto. Allegare un altro siringa a questa parte dell&#39;ago emulsionante. </li><li> Passare la miscela attraverso l&#39;ago per un totale di 40 volte (20 volte avanti e indietro) per trasformare le cercarie. </li><li> Posizionare le siringhe verticalmente in modo tale che la miscela è contenuta nella siringa in basso e in alto la siringa è vuota. Rimuovere la siringa superiore e disinfettare in etanolo al 70%. </li><li> Deposito goccia a goccia miscela in un ambiente pulito ad alta parete 100 millimetri x 50 millimetri piatto pirex o un piatto cristallizzazione. Disinfettare di aghi e siringhe in etanolo al 70%. </li><li> Aggiungi 37 ° C RPMI 1640 completo dei media in modo che ilparte inferiore della scatola di Petri è completamente coperto, come se versando un piatto di brodo Luria. </li><li> Agitare delicatamente per raccogliere schistosomules al centro della scatola di Petri. Evitare gli schizzi. Nota: Può essere utile per spegnere la luce cappa per questa fase di visualizzare in modo più adeguato la schistosomules al centro. </li><li> Immediatamente deposito schistosomules concentrata in un nuovo 12-pozzetti di coltura cellulare e con un contagocce sterile. </li><li> Ripetere 2.9 e 2.10 fino schistosomules non sono più presenti nel centro del piatto (circa 10 volte). Deposito ogni successiva raccolta in un pozzo fresco. </li><li> Riempire ogni bene fino a metà corsa (~ 1,5 ml) con i media preriscaldata RPMI completo. </li><li> Visualizzare su un microscopio invertito per verificare la presenza di schistosomules e assenza di cercarie. </li><li> Crescere il schistosomules in un incubatore CO 2 set a 37 ° C e 5% di CO 2. </li></ol> 3. Rappresentante dei risultati: In attesa di trasferimento schistosomules trasformato in terreni di coltura, la visualizzazione sotto una rovesciata luminoso microscopio a campo (vedere la tabella attrezzature per un esempio) dovrebbe produrre una immagine simile alla figura 1, in cui solo schistosomules trasformato sono presenti nel pozzo. Potenziali contaminanti potrebbero includere cercarie intatta, code mozzate cercarie, o altri residui. Se non microscopio invertito è disponibile, un microscopio standard e persino un campo di applicazione dissezione sarà sufficiente visualizzare la schistosomules. Si noti che questa procedura non sempre trasformare il 100% delle cercarie. <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="Figura 1"/> Figura 1: Schistosomulum Trasformato <img src="/files/ftp_upload/3191/3191fig2.jpg" alt="Figura 2"/> Figura 2: Cercariae e Tails Severed

<ol>
	<li>Ross, A. G., Bartley, P. B., Sleigh, A. C., Olds, G. R., Li, Y., Williams, G. 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. H., Dickman, K., Tisch, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reassessment+of+the+cost+of+chronic+helmintic+infection:+a+meta-analysis+of+disability-related+outcomes+in+endemic+schistosomiasis.">Reassessment of the cost of chronic helmintic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis.</a> Lancet. 365, 1561-159 (2005).</li><li>Haas, W., Grabe, K., Geis, C., Pach, T., Stoll, K., Fuchs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+and+invasion+of+human+skin+by+Schistosoma+mansoni+cercariae:+the+key-role+of+L-arginine.+Parasitology.">Recognition and invasion of human skin by Schistosoma mansoni cercariae: the key-role of L-arginine. Parasitology.</a> 124, 153-167 (2002).</li><li>Grabe, K., Haas, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigation+within+host+tissues:+cercariae+orientate+towards+dark+after+penetration.">Navigation within host tissues: cercariae orientate towards dark after penetration.</a> Parasitol Res. 93, 111-113 (2004).</li><li>Shiff, C. J., Cmelik, S. H., Ley, H. E., Kriel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+human+skin+lipids+on+the+cercarial+penetration+responses+of+Schistosoma+haematobium+and+Schistosoma+mansoni.">The influence of human skin lipids on the cercarial penetration responses of Schistosoma haematobium and Schistosoma mansoni.</a> J Parasitol. 58, 476-480 (1972).</li><li>Saladin, K. S. <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:+cercarial+responses+to+irradiance+changes.">Schistosoma mansoni: cercarial responses to irradiance changes.</a> J Parasitol. 68, 120-124 (1982).</li><li>Chai, M., McManus, D. P., McInnes, R., Moertel, L., Tran, M., Loukas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+lung+schistosomula,+in+vitro+cultured+schistosomula+and+adult+Schistosoma+japonicum.">Transcriptome profiling of lung schistosomula, in vitro cultured schistosomula and adult Schistosoma japonicum.</a> Cell Mol Life Sci. 63, 919-929 (2006).</li><li>Gobert, G. N., Tran, M. H., Moertel, L., Mulvenna, J., Jones, M. K., McManus, D. 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>

Nessun conflitto di interessi dichiarati.

Diversi potenziali problemi potrebbero insorgere durante la procedura di trasformazione, tuttavia, questo protocollo è stato progettato per evitare la maggior parte di questi problemi. Un potenziale pericolo è l&#39;intasamento dell&#39;ago emulsionante, che nasce di solito a causa di detriti lasciati dalla raccolta al punto 1.3. Assicuratevi di lasciare tutto il particolato di stabilirsi a passo 1,5 e di trasferire le cercarie attenzione al punto da 1,6 a evitare questo problema. Inoltre, come descritto in precedenza, lavorando all&#39;interno di una cappa di sicurezza biologica, l&#39;uso di dispositivi di protezione individuale (anche visiere) e di evitare una pressione eccessiva sulla siringhe è consigliabile. Staccata code cercarie possono essere visualizzati nei media, che è indicativo della scarsa separazione durante la fase 2.9. Le code mostra uno spasmo, come movimento e hanno una forma distinta (vedi Figura 2), e questa convenzione sono facilmente distinguibili dal schistosomules. La coda può essere rimosso lavando il schistosomules in eccesso di RPMI completo che permette la schistosomules di stabilirsi. Le code rimarranno galleggianti e possono essere aspirate fuori. Se cercarie intatti sono presenti nei media, la trasformazione in fase 2.5 non è stato eseguito correttamente. L&#39;immagine di un cercarie intatto è raffigurato in Figura 3b di riferimento. Se si verifica questo problema, assicurarsi che l&#39;ago emulsionante utilizzato è delle dimensioni appropriate. Inoltre, il numero di volte in cui viene passato il composto cercarie attraverso l&#39;ago può essere aumentata, anche se 40 volte è in genere molto più che sufficiente per raggiungere il 100% di taglio delle code cercarie. Trasformazione di cercarie in schistosomules è stato descritto nel 1974 da Colley e Wikel (13). Da allora, la preparazione di schistosomules è stato fatto utilizzando la strategia di taglio con un ago e la siringa come descritto in questo protocollo, o utilizzando un approccio vortex e separati con gradienti di Percoll (14, 15) o vorticoso, come dimostrato in questo documento. Queste due tecniche per la preparazione schistosomules sono ben descritti da Fred Lewis (16) e ulteriori informazioni per la coltura di ulteriori schistosomules per la crescita a lungo termine si possono trovare presso il Centro Risorse NIAID schistosoma ( <a href="http://www.schisto-resource.org/">www.schisto-resource.org</a> ).

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> 22g x 2-7/8 &quot;ago emulsionante </td><td> Fisher Scientific </td><td> 14-825-17G </td></tr><tr><td> RPMI 1640 Media 1X (500ml) </td><td> Invitrogen </td><td> 11835030 </td></tr><tr><td> Siero fetale bovino (FBS) inattivato con il calore </td><td> Invitrogen </td><td> 10082139 </td></tr><tr><td> Penicillina / streptomicina (100X) </td><td> Invitrogen </td><td> 15070063 </td></tr><tr><td> Piatto cristallizzando (100mm) </td><td> Fisher Scientific </td><td> 08-741D </td></tr><tr><td> Pinzetta General Purpose </td><td> Fisher Scientific </td><td> 10-316C </td></tr><tr><td> Petco Acquario netto artemia </td><td> Petco </td><td> Codice: 1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

Parassiti schistosoma sono gli agenti causali della schistosomiasi, una malattia cronica debilitante che colpisce oltre 200 milioni di persone nel mondo e al secondo posto alla malaria tra le malattie parassitarie, in termini di salute pubblica e impatto socio-economico (1-4). Schistosoma sono vermi parassiti trematode con un ciclo di vita complesso interscambio tra una vita parassitaria in ospiti di molluschi e di mammiferi con intervenendo libero nuoto fasi. In breve, nuoto libero cercarie infettare un ospite mammifero penetrando la pelle con l&#39;ausilio di proteasi secrete, durante i quali le cercarie perdono la coda, si trasforma in schistosomules. Il schistosomules deve ora eludere il sistema immunitario ospite, sviluppare un intestino per la digestione dei globuli rossi, e migrano attraverso i polmoni e la circolazione portale in rotta verso la destinazione finale nel sistema portale epatico ed eventualmente le vene mesenteriche (per S. mansoni) dove maschile e femminile coppia vermi e compagno, producendo centinaia di uova ogni giorno. Alcune delle uova sono espulso dal corpo in acqua dolce, dove le uova si schiudono in free-nuoto miracidi (5-10). Il miracidi infettare specie di lumaca specifici e si trasformano in sporocisti madre e figlia, che a loro volta, producono cercarie infettive, completando il ciclo di vita. Purtroppo, l&#39;intero ciclo di vita schistosoma non può essere coltivato in vitro, ma cercarie infettive può essere trasformato in schistosomules, e il schistosomules può essere coltivato per settimane per l&#39;analisi dello sviluppo schistosoma in vitro o l&#39;analisi microarray. In questo protocollo, forniamo una descrizione visiva di trasformazione cercarie e in coltura in vitro di schistosomules. Noi capannone cercarie infettive dal glabrata Biomphalaria lumaca di accoglienza e manualmente li trasformano in schistosomules staccando la coda con un emulsionante doppio attacco ago. In vitro cercarie trasformazione e tecniche di coltura schistosomules descritte evitare l&#39;uso di un ospite mammifero, che semplifica la visualizzazione di schistosomi e facilita la raccolta del parassita per l&#39;analisi sperimentale. Trasformazione in vitro e le tecniche di coltura schistosomi sono state fatte da anni (11, 12), ma non esistono ancora protocolli visivo sono stati sviluppati che sono disponibili per l&#39;intera comunità.

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

Ci descrivono un<em&gt; In vitro</emMetodo&gt; per schistosomula coltura del parassita verme piatto<em&gt; Schistosoma mansoni</em&gt;, Attraverso la raccolta e la trasformazione di cercarie infettive da acqua dolce glabrata ospite intermedio Biomphalaria lumaca.

Trasformazione e cercarie In vitro La coltivazione di Schistosoma mansoni Schistosomules

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

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Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

18.0K Views.  Case Western Reserve University. Ci descrivono un In vitro per schistosomula coltura del parassita verme piatto Schistosoma mansoni, Attraverso la raccolta e la trasformazione di cercarie infettive da acqua dolce glabrata ospite intermedio Biomphalaria lumaca.

Video: 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.

Con ovuli<em&gt; Schistosoma mansoni</em&gt; Come<em&gt; In vivo</em&gt; Modello di elminti-indotta infiammazione polmonare

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

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Immunologia e infezioni

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.

Massa isolamento e coltivazione In Vitro delle fasi Intramolluscan del trematode umano sangue 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 dello sviluppo

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.

Trasduzione transitoria delle forme strobilate di Echinococcus granulosus

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

Genetica

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.

Uso di<em&gt; Shigella flexneri</em&gt; Per studiare le interazioni autofagia-Citoscheletro

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 caratterizzazione di vescicole extracellulari da adulto 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.

Generazione di transgenici e knockout in specie di Strongyloides mediante microiniezione

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.

Generazione di sporozoiti geneticamente modificati di 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 captazione delle cellule dendritiche di piccole vescicole extracellulari da 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.

Nucleofezione e Propagazione In Vivo dei Parassiti dell'Emeria del Pollo

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.

Trasformazione e cercarie In vitro La coltivazione di Schistosoma mansoni Schistosomules

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