Questo lavoro è stato sostenuto dal National Institutes of Health (NIH) AI091759 e il morbo di Crohn e Colite Fondazione William America e Shelby Modell Award Famiglia Research Foundation (a MN). Schistosoma topi infettati sono stati forniti dal Centro NIAID Resource schistosoma (NIAID Contract N01 A130026).

1. Purificante Schistosoma mansoni Eggs <ol><li> Infect svizzero-Webster topi con cercariae schistosoma, che è lo stadio infettivo del ciclo di vita Schistosoma 8. In alternativa, ottenere schistosoma topi infettati dal Resource Center NIAID schistosomiasi ( <a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/~~V</a> ). Lasciare topi di 6-7 settimane dopo l&#39;infezione, per cui il tempo-punto, le uova sono presenti nel fegato e può essere recuperato come descritto di seguito (vedi Fig. 1). </li><li> Euthanize topi con CO 2, e bagnare con il 70% di etanolo. Posizionare il mouse sul retro. Utilizzare pinze smussate e forbici per tagliare via la pelle e del peritoneo sottostante. Excise il fegato, che si trova sotto la gabbia toracica e del diaframma. </li><li> Finemente macinata fegati e aggiungere 20 ml per fegato di miscela di digestione, composta di collagenasi / dispasi (0,5 mg / ml), penicillina (100 U / ml) e streptomicina (100 ug / ml) in PBS. Mettere MIXTure in una provetta da 50 ml Falcon ed incubare una notte a 37 ° C shaker. </li><li> Centrifugare miscela a 400 xg / 3 min / 20 ° C. Delicatamente versare il liquido in eccesso e riempire tubo con PBS. Centrifugare e ripetere lavaggio PBS. Dopo l&#39;ultima centrifugazione, Risospendere il pellet in 25 ml di PBS. </li><li> Filtrare il sedimento risospeso attraverso un filtro metallico standard di cucina (usare una siringa da 50 ml stantuffo per schiacciare i pezzi di fegato attraverso in un bicchiere di vetro), seguita da passare la miscela attraverso un setaccio fine (utilizzare siringa da 10 ml per spingere miscela attraverso il setaccio fine in un bicchiere di vetro secondo). </li><li> Mettere impasto strained in una provetta da 50 ml Falcon. Centrifugare a 400 xg / 5 min / 20 ° C. Versare con cautela out surnatante senza perdere pellet. Risospendere il pellet in 3 ml di PBS. </li><li> Overlay mescolare delicatamente su un Percoll 20%, 40 mL di gradiente Percoll 8 ml e 32 ml di saccarosio 0,25 M (FW = 342,3 così 8,56 g sucrose/100 mL H 2 0). </li><li> Centrifugare 10 min / 800 xg / 20 ° C. Pipettare fuori unod scartare gelatinoso strato superiore delle cellule epatiche. Rimuovere uovo pellet in basso con una pipetta di trasferimento e messo in una provetta da 15 ml Falcon. </li><li> Wash 3X pellet in 10 ml di PBS / EDTA 1 mM / 1 mm impostazioni centrifuga EGTA utilizzando: 3 min / 30 xg / 20 ° C. Risospendere in 500 pl PBS. </li><li> Mix Overlay su una nuova Percoll 25%, 10 mL gradiente (2,5 ml di Percoll e 7,5 mL saccarosio 0,25 M) utilizzando le impostazioni centrifuga: 10 min / 800 xg / 20 ° C. </li><li> Lavare 3 volte in 10 ml di PBS utilizzando le impostazioni di centrifuga: 3 min / 30 xg / 20 ° C. </li><li> Negli ultimi 10 mL di lavaggio, rimuovere 100 ul per contare le uova. Uova risolvere rapidamente, in modo che il tubo Falcon devono essere miscelati bene prima di rimuovere campione. Diluire il campione da 100 ml con 900 microlitri di PBS. 100 microlitri della miscela dovrebbe essere considerato come gocce su un vetrino da microscopio utilizzando un microscopio da dissezione (diluizione 1:10). Wide-foro puntali devono essere utilizzati. </li><li> Risospendere uova a 50.000 uova / ml in PBS. Le uova possono essere conservati per alcuni mesi a -80 ° C e scongelato una o TWICe per l&#39;uso. Prima dell&#39;uso, ispezionare sotto un microscopio per garantire che le uova sono ancora intatte (vedi fig. 1). </li></ol> 2. Sensibilizzazione intraperitoneale con S. Uova mansoni: Giorno 0 <ol><li> Preparare le uova a 5.000 pl eggs/100 PBS in una provetta da 5 mL snapcap. Per l&#39;iniezione, stimano uova 50% più del necessario. </li><li> Caricare un ago da 23 gauge 3/4inch e una siringa da 1 ml con 100 microlitri di sospensione uovo al mouse. Uova risolvere, in modo da aghi di carico con le uova immediatamente prima dell&#39;utilizzazione. </li><li> Roccia siringa avanti e indietro per mescolare le uova prima di ogni iniezione, e iniettare per via intraperitoneale 100 microlitri di sospensione per mouse. </li></ol> 3. Retro-orbitale sfida endovenosa con S. Uova mansoni: Giorno 14 <ol><li> Preparare le uova di cui sopra a 5.000 eggs/100 pl. </li><li> Anestetizzare topi in camera specializzata con isofluorano (4%), o intraperitoneale con xilazina (10 mg / kg) e ketamina (120 mg / kg). Profondità di Anesthesia è determinato dal pizzicamento della zampa e garantendo l&#39;assenza di risposta fisica. </li><li> Dopo il caricamento della siringa da 1 ml e 23 gauge 3/4inch con uova, utilizzare forcipe per piegare l&#39;ago con un angolo di 90 ° con la smussatura rivolta verso il basso l&#39;angolo. Assicurarsi che non siano presenti bolle. </li><li> Posizionare il mouse su un lato. Ritrarre la pelle in modo da sporgere occhio, e iniettare 100 microlitri nei vasi dietro il bulbo oculare in un angolo di 45 ° al naso. </li><li> Ritirare l&#39;ago ed applicare una leggera pressione per controllare l&#39;emorragia. </li></ol> Tutte queste procedure deve essere eseguita con cura, e topi deve essere monitorata fino recupero dall&#39;anestesia. Questi protocolli sono stati approvati dalla University of Pennsylvania Institutional Animal Care e del Comitato uso (IACUC). 4. Sperimentale Vendemmia: Day 22 <ol><li> Euthanize topi con CO 2. </li><li> Bagnare con il mouse etanolo al 70%, e il luogo sul retro. </li><li> Utilizzando blunt forceps, afferrare l&#39;addome del mouse e fare una piccola incisione nella pelle. Rimuovere con cura la pelle con le forbici ed esporre la gabbia toracica e del peritoneo. </li><li> Tagliare il peritoneo. Tagliare il aorta addominale di recuperare il sangue con una pipetta Pasteur. Conservare su ghiaccio. </li><li> Spostare il fegato giù per esporre il diaframma. Usare le forbici per tagliare sottili il diaframma. Tagliare con cautela aperta la gabbia toracica per esporre il tessuto polmonare. </li><li> Recuperare i linfonodi che drenano parathymic il polmone con una pinza fine. Questi sono infilato sotto la gabbia toracica, su entrambi i lati del timo 9. Conservare il ghiaccio in 1mL mezzi sterili (DMEM supplementato con 10% inattivato al calore siero di vitello fetale, 100 U / ml penicillina, 100 pg / ml streptomicina, 2 mM L-glutammina, 50 pM 2-mercaptoetanolo, tutti disponibili da Invitrogen). </li><li> Per recuperare il BAL, e gonfiare i polmoni per l&#39;analisi istologica, l&#39;intubazione endotracheale deve essere eseguita. Esporre la trachea spostando le ghiandole salivari di lato, e placing pinza sottile sotto la trachea. Utilizzando forbici sottili, tagliare un foro al centro della trachea, e inserire tubi nei polmoni. Fissare tubi legando con filo chirurgico. </li><li> Fissare PBS-riempita siringa da 1 ml al tubo e gonfiare i polmoni con 1 ml di PBS. Lavare accuratamente recuperare BAL con la siringa. Conservare su ghiaccio. </li><li> Attaccare 4% paraformaldeide (PFA) in PBS-riempita siringa da 1 ml per il tubo e gonfiare i polmoni. </li><li> Rimuovere il tubo, e serrare il filo chirurgico per prevenire le infiltrazioni della PFA. </li><li> Analizzare attentamente le tessuto polmonare e posto in una provetta Falcon 50 ml con 5 ml 4% PFA. </li></ol> 5. Preparazione dei tessuti recuperati <ol><li> Siero: memorizzazione seguito su ghiaccio per 30 minuti a 2 ore alla coagulazione del sangue, siero viene recuperato dal sangue mediante centrifugazione (13000 xg / 10 min / 4 ° C). Il siero è conservato a -20 ° C e può essere utilizzato per l&#39;ELISA citochine o S. mansoni uovo antigene-specifica isotipo IgG ELISA <sup&gt; 6, 10. </li><li> Linfonodi: sospensioni di cellule singole sono preparati e conta delle cellule utilizzate per valutare l&#39;entità della risposta immunitaria. Per esaminare la polarizzazione delle cellule Th2, le cellule vengono ri-stimolate con S. mansoni antigene uovo per 72 ore. Colorazione Intracellar citochina può essere esaminata mediante citometria di flusso, e supernatanti può essere recuperato e conservato a -20 ° C per ELISA di citochine Th2 (IL-4, IL-5, IL-13) 6. </li><li> BAL: Le cellule sono pellettizzato (400 xg / 5 min / 4 ° C). BAL viene recuperato e conservato a -20 ° C per l&#39;analisi di citochine Th2 mediante ELISA. BAL conteggi celle vengono utilizzate come un visualizzatore per infiammazione delle vie aeree. Preparazioni di citocentrifuga 10,000-100,000 cellule (500 giri / min 5/4 ° C), seguita da colorazione Diffquik consentirà enumerazione di infiltrato infiammatorio 4, 6. In alternativa, le cellule BAL può essere esaminata mediante citometria di flusso 6. </li><li> Tessuto polmonare: il tessuto polmonare è stored notte a 4 ° C. Tessuto fissato è incluso in paraffina, sezionati e montati su vetrini per l&#39;analisi istologica ed immunofluorescenza. </li></ol> 6. Risultati rappresentativi Questo protocollo dettagli tutti i passaggi necessari per (i) la preparazione purificata Schistosoma mansoni uova per uso in vivo (Fig. 1), (ii) sensibilizzare e sfidare i topi con queste uova e (iii) recuperare gli organi per l&#39;esame della risposta infiammatoria del polmone. Questo modello in vivo guida riproducibile una potente risposta infiammatoria polmonare Th2. Ciò è dimostrato da infiammazione delle vie aeree e come eosinofilia visualizzati tramite conta delle cellule del BAL e preparati citocentrifuga (Fig. 2). H &amp; E-colorate sezioni polmonari possono essere esaminati per l&#39;egg-indotta granulomi (Fig. 3a), e la colorazione di immunofluorescenza delle sezioni polmonari permette la visualizzazione di macrofagi attivati ​​alternativamente e Th2 citochine indotte geni come resistenzatin-come molecola (RELM) α (Fig. 3b). L&#39;antigene-specifica risposta citochina Th2 CD4 viene esaminata da ELISA per IL-5, IL-4 e IL-13-antigene di cellule stimolate parathymic linfonodi (Fig. 4). <img alt="Figura 1" src="/files/ftp_upload/3905/3905fig1.jpg" /> Figura 1 Schistosoma mansoni modello iniezione di uova foto rappresentativi di fegato granulomatose, caratteristiche di alta S.mansoni-uovo oneri, e di uova S.mansoni sono mostrati -.. Dimensione media (L) 120 micron (da W) 50 pm. <img alt="Figura 2" src="/files/ftp_upload/3905/3905fig2.jpg" /> Figura 2. S. mansoni iniezione di uova guida infiammazione delle vie aeree. A. Numero di cellule BAL (x10 5) da naïve (N) o S.mansoni (Sm) uovo iniettato nei topi. * P &lt;0,05. B. citocentrifuga preparazione di cellule BAL. Mac, macrofagi; Eos, eosinofili, LINFA, linfociti. Bar, 10 micron. <img alt="Figura 3" src="/files/ftp_upload/3905/3905fig3.jpg" /> Figura 3. Visualizzazione di granulomi polmonari e macrofagi attivati ​​alternativamente che circondano l&#39;uovo S.mansoni. A. Granuloma polmonare che circonda l&#39;uovo Sm (freccia nera) è stata visualizzata in H &amp; E-colorate sezioni polmonari. B. colorazione per immunofluorescenza RELMα (verde), recettore del mannosio (rosso) e DAPI (blu) rivela in alternativa macrofagi attivati ​​(freccia bianca) nel granuloma che circonda la autofluorescente Sm uovo (freccia nera). Bar, 50 pm. <img alt="Figura 4" src="/files/ftp_upload/3905/3905fig4.jpg" /> Figura 4. S.mansoni uovo antigene-specifica risposta delle citochine Th2. Drenante parathymic cellule linfonodi di topi naïve (N) o uova Sm-iniettata sono state stimolate con Sm &lt;/ Em&gt; antigene uovo per 72 ore, seguita da ELISA di supernatanti per IL-4, IL-5 e IL-13. Scala, ng / mL. * P &lt;0,05.

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	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis:+report+of+a+WHO+expert+committee.">Prevention and control of schistosomiasis and soil-transmitted helminthiasis: report of a WHO expert committee.</a> WHO Technical Report Series 912. , (2002).</li><li>Pearce, E. J., MacDonald, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+schistosomiasis.">The immunobiology of schistosomiasis.</a> Nat. Rev. Immunol. 2, 499-511 (2002).</li><li>Sandler, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+profiles+during+acute+pathogen-induced+pulmonary+inflammation+reveal+divergent+roles+for+Th1+and+th2+responses+in+tissue+repair.">Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and th2 responses in tissue repair.</a> J. Immunol. 171, 3655-3667 (2003).</li><li>Perrigoue, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-31-IL-31R+interactions+negatively+regulate+type+2+inflammation+in+the+lung.">IL-31-IL-31R interactions negatively regulate type 2 inflammation in the lung.</a> J. Exp. Med. 204, 481-487 (2007).</li><li>Sandor, M., Weinstock, J. V., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulomas+in+schistosome+and+mycobacterial+infections:+a+model+of+local+immune+responses.">Granulomas in schistosome and mycobacterial infections: a model of local immune responses.</a> Trends Immunol. 24, 44-52 (2003).</li><li>Nair, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatively+activated+macrophage-derived+RELM-{alpha}+is+a+negative+regulator+of+type+2+inflammation+in+the+lung.">Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung.</a> J. Exp. Med. 206, 937-952 (2009).</li><li>Dalton, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+schistosome+eggs+and+miracidia+free+of+contaminating+host+tissues.">A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.</a> Parasitology. 115, 29-32 (1997).</li><li>Lewis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+laboratory+maintenance+of+Schistosoma+mansoni,+with+observations+on+three+schistosome/snail+host+combinations.">Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations.</a> J. Parasitol. 72, 813-829 (1986).</li><li>Tilney, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+lymphatic+drainage+in+the+adult+laboratory+rat.">Patterns of lymphatic drainage in the adult laboratory rat.</a> J. Anat. 109 (Pt. 3), 369-383 (1971).</li><li>Lewis, F., Coligan, J. E. <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> Current protocols in immunology. Chapter 19, Unit 19 (2001).</li><li>Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2+cells+are+required+for+the+Schistosoma+mansoni+egg-induced+granulomatous+response.">Th2 cells are required for the Schistosoma mansoni egg-induced granulomatous response.</a> J. Immunol. 160, 1850-1856 (1998).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrotic+disease+and+the+T(H)1/T(H)2+paradigm.">Fibrotic disease and the T(H)1/T(H)2 paradigm.</a> Nat. Rev. Immunol. 4, 583-594 (2004).</li><li>Cheever, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+hepatic+fibrosis+and+granuloma+size+among+mouse+strains+infected+with+Schistosoma+mansoni.">Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni.</a> Am. J. Trop. Med. Hyg. 37, 85-97 (1987).</li></ol>

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Qui, un modello che impiega le uova Schistosoma mansoni di indurre l&#39;infiammazione polmonare di tipo 2 viene descritto. Tratti caratteristici di questo modello includono il potente risposta immunitaria Th2, infiammazione delle vie aeree e la formazione di granuloma polmonare. Dal momento che questi parametri dipendono le risposte delle cellule CD4 Th2 11, questo è un modello utile per studiare gli effetti delle cancellazioni proteine ​​specifiche o lineage-specifica sulle risposte delle cell...

<table border="1"><tbody><tr><td> Reagente </td><td> Dimensione / Descrizione </td><td> Azienda </td><td> Catalogo # </td></tr><tr><td> Collagenasi / dispasi </td><td> 500 mg </td><td> Roche Applied Science </td><td> 11 097 113 001 </td></tr><tr><td> Diff Quik Hema 3 Stain </td><td> Imballare </td><td> Fisher Scientific Co. LLC </td><td> 122-911 </td></tr><tr><td> DMEM </td><td> Liquido </td><td> Gibco </td><td> 11965 </td></tr><tr><td> Falcon 50mL </td><td> Imballare </td><td> Falco </td><td> 352070 </td></tr><tr><td> Intramedic PE Tubi </td><td> 0,58 millimetri </td><td> Becton Dickinson and Co. </td><td> 427410 </td></tr><tr><td> Isoflurano </td><td> 250 mL </td><td> Abate Animal Health </td><td> 52600405 </td></tr><tr><td> Ketamina </td><td> 100 mg / mL </td><td> Fort Dodge, la salute degli animali </td&gt; <td> N / A </td></tr><tr><td> Recettore anticorpo mannosio </td><td> Biotina </td><td> ABD Serotec </td><td> MCA2235B </td></tr><tr><td> Metallic Filtro </td><td> Cucina filtro standard </td><td> Obiettivo </td><td> Laurentiis </td></tr><tr><td> Paraformaldeide </td><td> 16% </td><td> Electron Microsc. Scienze </td><td> 15710 </td></tr><tr><td> Penicillina / streptomicina </td><td> 100x </td><td> Gibco </td><td> 15070 </td></tr><tr><td> Percoll </td><td> 1 litro </td><td> GE Healthcare Biosciences </td><td> 17 01 0891 </td></tr><tr><td> Anticorpi RELMα </td><td> 50 ug </td><td> Peprotech </td><td> 500-P214 </td></tr><tr><td> Filo chirurgico </td><td> 2,0 USP 100 yds </td><td> Surgical Specialties Corp. </td><td> SP118 </td></tr><tr><td> Siringa con ago </td><td> 23G, 3/4 di pollice </td><td> BD Vacutainer Lab. Med. </td><td>305143 </td></tr><tr><td> USA Standard Testing Sieve </td><td> 150 pm, 0.0059 &quot; </td><td> WS Tyler, Inc. </td><td> ASTME-11, Spec. No. 100 </td></tr><tr><td> Xilazina </td><td> 20 mg / mL </td><td> Maggiordomo </td><td> N / A </td></tr></tbody></table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

Parassiti Schistosoma sono trematodi del sangue che infettano circa 200 milioni di persone in tutto il mondo 1. In infezione cronica con Schistosoma, la patologia grave, inclusa la fibrosi epatica e splenomegalia, è causata dalla risposta immunitaria alle uova di parassiti piuttosto che il parassita stesso 2. Uova Parasite indurre una risposta Th2 caratterizzata dalla produzione di IL-4, IL-5 e IL-13, l&#39;attivazione dei macrofagi alternativa e il reclutamento di eosinofili. Qui, descriviamo l&#39;iniezione di uova di Schistosoma mansoni come un modello per esaminare parassita risposte specifiche citochine Th2 nei polmoni e dei linfonodi drenanti, la formazione di granulomi polmonari che circondano l&#39;uovo e l&#39;infiammazione delle vie aeree. Dopo sensibilizzazione intraperitoneale e sfida endovenosa, S. mansoni uova vengono trasportati al polmone attraverso le arterie polmonari in cui sono intrappolati all&#39;interno del parenchima polmonareda granulomi composti da linfociti, eosinofili e macrofagi attivati ​​alternativamente 3-6. Associata alla formazione di granulomi, infiammazione nelle bronco-alveolari spazi, l&#39;espansione dei linfonodi drenanti e l&#39;attivazione delle cellule T CD4 si può osservare. Qui dettaglio il protocollo per isolare le uova Schistosoma mansoni da fegati infettati (modificato da 7), la sensibilizzazione e sfidando i topi, e il recupero degli organi (lavaggio bronco-alveolare (BAL), del polmone e linfonodi drenanti) per l&#39;analisi. Abbiamo anche istologico dati rappresentativi e immunologici e suggerimenti per l&#39;analisi immunologica aggiuntivo. In generale, questo metodo fornisce un modello in vivo per indagare elminti indotte risposte immunologiche nel polmone, che è ampiamente applicabile allo studio di Th2 malattie infiammatorie comprese le infezioni elminti, malattie fibrotiche, infiammazione allergica e asma. I vantaggi di questo modello per lo studio del tip2 e infiammazione nel polmone includono la riproducibilità di una potente risposta infiammatoria Th2 nei polmoni e linfonodi drenanti, la facilità di valutazione di infiammazione mediante esame istologico dei granulomi circondano l&#39;uovo, e il potenziale di conservazione a lungo termine del parassita uova.

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

Schistosoma mansoni uova sono potenti stimolatori del T helper di tipo 2 (Th2) risposta immunitaria, caratteristica di infezioni parassitarie, asma e infiammazione allergica. Questo protocollo utilizza S. mansoni uovo iniezione per generare un CD4 Th2 citochine indotta da risposta infiammatoria nel polmone, caratterizzata dalla formazione di granulomi intorno al polmone l&#39;uovo, eosinofilia e l&#39;attivazione dei macrofagi alternative.

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

13.7K Views.  University of Pennsylvania. Schistosoma mansoni uova sono potenti stimolatori del T helper di tipo 2 (Th2) risposta immunitaria, caratteristica di infezioni parassitarie, asma e infiammazione allergica. Questo protocollo utilizza S. mansoni uovo iniezione per generare un CD4 Th2 citochine indotta da risposta infiammatoria nel polmone, caratterizzata dalla formazione di granulomi intorno al polmone l'uovo, eosinofilia e l'attivazione dei macrofagi alternative.

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

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.

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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Immunologia e infezioni

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Induzione di Graft-versus-host disease e<em&gt; In Vivo</em&gt; T monitoraggio cellulare Utilizzando un MHC-abbinato modello murino

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo metodo di imaging per distinguere infiammazione acuta e cronica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Un<em&gt; Ex vivo</em&gt; Modello di un attacco T-Cell Oligodendrocyte-diretto nel acute cervello fette

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza Virus Propagazione in embrionate di pollo Uova

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Un metodo ottimizzato per l&#39;isolamento ed espansione invariante Natural Killer cellule T da Spleen mouse

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Sonde ottiche Screening di nuovi batteri specifici sul tessuto polmonare umano Ex Vivo di endomicroscopia confocale Laser

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Valutazione dei citotossici e gli effetti immunomodulatori di sostanze in fette di polmone umano di taglio di precisione

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

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 ...