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 ...

cercarial-transformation-vitro-cultivation-schistosoma-mansoni

Research

JoVE Journal

Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

17.9K 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

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.

In uncoating vitro di 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 ...

Ricerca

Immunologia e infezioni

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 ...

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.

Quantitative<em&gt; In vitro</em&gt; Assay misurare Adesione neutrofili attivati ​​per primarie umane endoteliali microvascolari in condizioni statiche

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.

Coltivazione di<em&gt; Heligmosomoides polygyrus:</em&gt; Un immunomodulante nematode parassita ei suoi prodotti secreti

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.

Trasformazione Biolistic di una fluorescente Tagged Gene nel Opportunistic Fungal patogeni<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.

Trasformazione di lievito probiotico e il loro recupero da gastrointestinali immunitario tessuti dopo sonda gastrica nei topi

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.

Metodi in vitro per il confronto obiettivo vincolante e CDC induzione Tra terapeutiche Anticorpi: Applicazioni nel Analisi 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 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 ...

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.

Analisi delle interazioni tra endobiotici e microbiota intestinale umana utilizzando sistemi di fermentazione bagno 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.

Trasfezione altamente efficiente dei macrofagi primari con mRNA trascritto 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 ...