We thank Sergio G&#243;mez-Medina for excellent technical support with mouse experiments. This work was supported by funds from the Leibniz Association and the Leibniz Center of Infection. A.L. is a recipient of a pre-doctoral fellowship from the Leibniz Graduate School.

Gli esperimenti sugli animali sono stati condotti secondo protocolli approvati e seguendo le linee guida della legge sulla protezione degli animali tedesca. Tutto il personale che effettuano esperimenti sugli animali passati programmi di formazione a seconda della categoria B o C della Federazione delle Associazioni scientifiche europee animali da laboratorio. 1. Generazione di ricombinante vivo attenuato Influenza vaccini da Reverse Genetics NOTA: Il protocollo dettagliato per la generazione di virus influenzali ricombinanti dalla genetica inversa è stata descritta da studi precedenti 5 ed è fuori dal campo di applicazione della presente relazione. In breve, il salvataggio di vaccini antinfluenzali adattate a freddo (CAV) è fatto in un armadio biosicurezza di classe II sotto il livello di biosicurezza 2 (BSL2) contenimento, e prevede passaggi descritti di seguito. Il protocollo di trasfezione e infezioni segue quella Martinez-Sobrido et al. 5. <ol><li> Generare un influenzale adattato freddo riassortantivaccino utilizzando come sfondo della adattato fredda ceppo A / Ann Arbor / 6/60 (H2N2), così come l&#39;emoagglutinina (HA) e neuraminidasi modificato (NA) del / PR / 8/34 (H1N1) A ( Figura 1A). La modifica nel gene NA è costituito da un sostituto basato su PCR di una sequenza conservata nel gambo della proteina (residui da 65 a 72) da una breve sequenza di cDNA codificante l&#39;ovalbumina di pollo (OVA) peptide -derived SIINFEKL (Figura 1B). Il peptide SIINFEKL è un peptide altamente immunodominante nel contesto della classe MHC H-2b risposta di topi C57BL / 6 12 I-ristretta. </li><li> Piatto 10 6 293T cellule per pozzetto in 6 pozzetti con DMEM al 10% di siero fetale bovino (FBS) supplementato con 1% di penicillina / streptomicina (P / E). </li><li> Preparare miscela di trasfezione plasmide: Aggiungere 1 ug di ciascuno dei plasmidi codificanti cDNA per PB2, PB1, PA, NP, M, NS dalla influenza A / Ann Arbor / 6/60 ceppo e 1 mg di plasmidi codificanti per la NA -SIINFEKL eHA da influenza A / PR / 8/34. Aggiungere pre-riscaldato OptiMEM (15 microlitri per un volume finale di 100 ul / pozzetto). </li><li> Incubare mix trasfezione per 30 minuti a RT. </li><li> Aggiungere 100 pl di miscela di trasfezione / pozzetto ed incubare per 16 ore a 37 ° C e 5% CO 2. </li><li> Cambiare supporti cella DMEM 0,3% BSA 1% P / E a 33 ° C e 5% CO 2 per 5 ore. </li><li> Aggiungere cellule MDCK permissive 10 6 influenzali in DMSO contenente 1 mg / ml di tripsina da pancreas bovino trattato con L-1-Tosylamide-2-feniletile clorometil chetone (TPCK-tripsina). Mantenere i 293T-MDCK co-culture per 2-3 giorni a 33 ° C e 5% di CO 2. </li><li> Raccogliere surnatanti di cellule co-culture MDCK-293T e chiaro per centrifugazione a 260 g per 5 min. </li><li> Inoculare 9-10 giorni di età uova embrionate di pollo in condizioni sterili. Per fare questo, fare un foro sulla parte superiore del guscio d&#39;uovo, come indicato da Martinez-Sobrido et al. 5. </li><li> Coprire il wit guscio d&#39;uovoh fuso cera con un batuffolo di cotone e incubare le uova di gallina inoculate per 3 giorni a 33 ° C. </li><li> Raccogliere il liquido allantoico dalle uova embrionate infette: Lavare i gusci d&#39;uovo con il 70% di etanolo. Aprire l&#39;uovo con una crepa molto delicatamente nella parte superiore e rimuovere la membrana allantoide con pinza sterile. Utilizzare una pipetta 10 ml di raccogliere quanto più fluido possibile (in genere 6 - 10 ml). Centrifugare a 260 xg per 10 min a 4 ° C e trasferire il fluido eliminato allantoico di nuovi tubi. </li></ol> Immunità 2. Monitoraggio vaccino specifico <ol><li> Tracciare delle cellule dendritiche peptide-cuscinetto (DC) <ol><li> Per la vaccinazione del mouse, anestetizzare i topi con il 5% isoflurano in ossigeno utilizzando un vaporizzatore di precisione. Utilizzare una pipetta 200 microlitri di somministrare 50 ml di PBS contenente 10 3 formanti placca unità (PFU) del vaccino esprimere il peptide SIINFEKL attraverso le goccioline direttamente alle narici del mouse. </li><li> Tre giorni dopo la vaccinazione, euthanize topi tramite anestesia isoflurano seguita da dislocazione cervicale. </li><li> Sollevare la pelle del mouse sulla linea mediana appena sotto la gabbia toracica con una pinza e tagliare una piccola incisione attraverso la pelle con le forbici. Dal punto di incisione, rendono un 3 - 5 cm di lunghezza tagliare attraverso la pelle verso la testa e la coda, poi due incisioni 2 cm dalla linea mediana lateralmente da ciascuna estremità. Staccare la pelle a rivelare la cavità peritoneale e toracica. Tagliare con cautela attraverso le membrane che circondano il peritoneo per esporre il torace. </li><li> Per accedere al mediastino tagliare il diaframma e la parte inferiore della gabbia toracica. Individuare e rimuovere 2 linfonodi mediastinici (mln), un po &#39;dorsale e laterale adiacente al timo, medialmente adiacente alla arteria anonima, nei pressi del lato ventrale della trachea. </li><li> Per raccogliere le mln utilizzano pinze curve. Posizionare pinze sotto i linfonodi e tirare delicatamente. Mettere i linfonodi in un 6-pozzetti contenente 1 ml di DMEM e rimuovere eventuali connective o tessuto adiposo che circonda il nodo. <ol><li> Tease bene ogni campione con due aghi 26 G collegati ad 1 ml siringhe. Per stuzzicare il linfonodo, tenere premuto con un ago mentre la rottura del linfonodo con l&#39;altro ago. Quando questo viene fatto correttamente, le cellule concentrate scoppiano dal linfonodo sono facilmente osservati nei media. Passare tutto il materiale tessuto attraverso un filtro di nylon 70 micron per ottenere sospensioni di cellule singole. </li></ol></li><li> Centrifugare sospensioni di cellule singole per 10 min a 260 xg in una centrifuga refrigerata. Risospendere il pellet di cellule in 2 ml di globuli rossi di lisi (rbcL) Buffer per lisare i globuli rossi. Lasciare lisi di procedere per 3 minuti a temperatura ambiente. </li><li> Neutralizzare tampone rbcL via centrifugazione e risospensione delle cellule in 2 ml di PBS ghiacciato. </li><li> Cellule piastra a forma di U piastre da 96 pozzetti ad una concentrazione di 10 7 cellule / ml in un volume finale di 100 ul. </li><li> Per la citometria a flusso di analisi, Cell Block Fc-recettori con1 mg di anticorpi CD16 / CD32-topo anti per 10 6 cellule per 15 min in ghiaccio. </li><li> Piatti centrifuga e scartano sovranatanti di piatto rapido passaggio. Incubare i pozzetti individuali con la combinazione di anticorpi superficie scelta. Tipicamente per la rilevazione di cellule dendritiche in linfonodi, il cocktail di anticorpi dovrebbe includere: 0,25 pg di anti-CD11c, 0,25 mg di CD11b, 0,5 mg di MHC di classe II, 0,25 mg di CD103 e 0,15 mg di CD8α in un volume finale di 100 ul. </li><li> Per gating negativo, aggiungere 0,25 mg di anti-CD3, CD19, NK1.1 o un lignaggio cocktail (Figura 2A). Strategie di gating alternativi e protocolli per una corretta compensazione di più coloranti fluorescenti sono disponibili sul sito web del Progetto Genoma immunologica. Oltre ai marcatori di superficie di scelta, aggiungere 1 mg di un anticorpo contro H-2K b legato alla SIINFEKL al cocktail. Questo anticorpo disponibile in commercio consente la visualizzazione delle DC in che il peptide SIINFEKL è legato alla superficie MHC di classe I. </li><li> Incubare le cellule con anticorpi di superficie per 30 min in ghiaccio. Proteggere piastra dalla luce. Piastre centrifugare a 260 xg per 5 minuti e lavare con 2 ml di PBS due volte. Trasferire le cellule di citometria a flusso tubi per l&#39;analisi. </li></ol></li><li> Valutazione del vaccino specifico la proliferazione delle cellule T <ol><li> Ottenere cellule T del donatore da disponibile commercialmente topi TCR-transgenici (topi OT-I) (C57BL / 6-Tg (Tcr aTcrb) 1100Mjb / J) in cui tutte le cellule T CD8 generate sono specifiche per il peptide SIINFEKL. Euthanize topi anestesia isoflurano seguita da dislocazione cervicale. Harvest milza effettuando un&#39;incisione nella cavità addominale. Posizionare milza / s in 2 ml di DMEM / milza e rimuovere il tessuto connettivo e grasso. </li><li> Generare sospensioni singola cella di milza seguendo la procedura descritta in 2.1.2 e 2.1.3. </li><li> Per arricchire ulteriormente la sospensione singola cella sulle cellule T, si applicano i protocolli di separazione tallone magneticoutilizzando sia le procedure di selezione positivi o negativi 12. Per differenziare donatore cellule riceventi, utilizzare i topi congenici modo che per esempio topi riceventi esprimono il CD45.1 marcatore leucociti mentre le cellule T del donatore esprimono CD45.2 12. </li><li> Per marcare le cellule T, incubare in 1 ml di PBS contenente 5 mM di carboxyfluorescein ester succinimidil (CFSE) in una densità cellulare ottimale di 5 x 10 6 cellule / ml. Incubare le cellule per 20 min a 37 ° C al riparo dalla esposizione alla luce. Dopo l&#39;incubazione, le cellule centrifugare a 260 xg per 5 minuti, il surnatante scarto, e risospendere le cellule in 3 ml di siero fetale bovino (FBS) per neutralizzare CFSE. </li><li> Cellule centrifugare a 260 xg durante 10 minuti e risospendere nel volume adeguato di PBS in modo che 100 ml di soluzione contiene 2 x 10 6 cellule. Infondere destinatario topi congenici con 2 x 10 6 cellule / topo via retro-orbitale sinusale iniezione 13. </li><li> Euthanize topi riceventi a giorni 3, 4 e 5 postvaccinazione in anestesia isoflurano seguita da dislocazione cervicale. Raccolto linfonodi mediastinici e preparare sospensioni singola cella per citometria a flusso, come indicato in 2.1.2-2.1.5. </li><li> Identificare le cellule del donatore dalla colorazione delle sospensioni di cellule singole a 100 l di un cocktail di anticorpi che contiene: 0,25 mg di anticorpi anti-topo CD3 CD4 e CD8; 0,5 mg di anti-topo CD45.1 o anticorpi CD45.2 (a seconda del fenotipo delle cellule del donatore). Lasciare la FL-1 canale del citometro a flusso aperto (FITC) per determinare il profilo di diluizione di CFSE 12 (Figura 2B). </li></ol></li></ol> 3. I topi chimerici per sezionare gene-specifica del vaccino Risposte NOTA: La generazione di concorrenza chimere di midollo osseo utilizzando modelli murini basati DTR permette la discriminazione di funzioni specifiche delle cellule durante la risposta immunitaria ai vaccini. Qui vi mostriamo una strategia aggiuntiva con la quale combinazione di DTR-esprimono donatore cells con celle di permessi topi knockout per studiare specifiche funzioni geniche durante l&#39;immunità in compartimenti cellulari specifici. Nell&#39;esempio generiamo topi con specifico delezione dei recettori toll-like 3 (TLR3) in Langerin + CD103 + DC. <ol><li> Preparazione delle cellule del donatore di midollo osseo <ol><li> Lavorare sotto un armadietto livello di biosicurezza II. Evitare la contaminazione utilizzando strumenti in autoclave esclusivamente. </li><li> Per generare midollo osseo topi chimerici, utilizzare CD45.1 congenic + C57BL / 6 topi femmina come destinatari, e utilizzare TLR3 - / -, Langerin-DTR / EGFP, o topi WT esprimono CD45.2 + come donatori. Utilizzando topi donatori e riceventi congenici permette la differenziazione delle cellule del donatore e del ricevente via citometria a flusso e questo permette attecchimento da valutare. </li><li> Per ottenere cellule del midollo osseo del donatore, eutanasia topi donatori con anestesia isoflurano seguita da dislocazione cervicale. Con forbici affilate eseguono incisioni intorno alle caviglie in modo che il mmuscolatura ouse è esposto. </li><li> Uso pinze smussato tirare la pelle del mouse e pelliccia delicatamente dalla caviglia verso i fianchi in modo che i muscoli delle gambe sono visibili. </li><li> Con le forbici affilate tagliare le gambe del mouse all&#39;altezza delle anche e sopra la testa del femore. </li><li> Posizionare gambe su una capsula di Petri e su ghiaccio. Lavorare velocemente e rimuovere tutti i muscoli per esporre le ossa. </li><li> Femori e tibie separati. Inserire ossa in una soluzione di etanolo al 70% per 5 minuti per rimuovere eventuali batteri e posto immediatamente su un&#39;altra capsula petri contenente 5 ml di DMEM privo di siero. Tagliare osso finisce con forbici affilate e lavare il midollo osseo nel DMEM. Per fare ciò, filo 1 ml di DMEM nell&#39;osso inserendo un ago 26 G attaccato ad una siringa da 5 ml. Assicurarsi che le ossa sembrano bianco (vuoto) dopo lavaggio. </li><li> Raccogliere tutte le cellule del midollo osseo di topi donatori in 10 ml di DMEM. Generare sospensioni singola cella di cellule del midollo osseo passando il raccolto attraverso un colino cella 70 micron. </li><li> Clorecchio sospensioni di cellule singole di cellule del midollo osseo per centrifugazione a 260 xg per 10 min a 4 ° C in una centrifuga refrigerata. Risospendere pellet cellulare in 10 ml di PBS. Deplezione delle cellule rosse del sangue utilizzando soluzione rbcL come descritto in 2.1.3 e 2.1.4. </li><li> Contare cellule del midollo osseo utilizzando un emocitometro o un contatore di cellule automatizzato. Valutare la vitalità cellulare del determinazione delle cellule morte con trypan colorazione blu 13. In modo ottimale, un animale produce singolo di circa 3 x 10 7 globuli bianchi / mouse con una redditività pari ad almeno l&#39;80%. </li><li> In caso di totale chimere del midollo osseo, preparare TLR-3 - cellule o peso del donatore - /. La concentrazione cellulare ottimale è di 2 x 10 7 cellule / ml da 2 x 10 6 cellule in 100 microlitri di PBS saranno utilizzati per il trapianto. Se non utilizzato immediatamente, conservare le cellule del donatore a -80 ° C utilizzando tecniche di congelamento lento e FBS con il 10% DMSO. Per il modello chimerico misto, mescolare le cellule Langerin-DTR-donatori e TLR3 - / - </sup&gt; Le cellule del donatore in un rapporto di 75:25. Preparare una miscela di 75: 25 Langerin-DTR: WT cellule del midollo osseo per generare topi di controllo. </li></ol></li><li> Irradiazione del mouse e trapianto <ol><li> Irradiare topi femmina tra 6-8 settimane di età in una fonte irradiatore cesio-137. Dare topi due dosi di 550 rad 4 ore di distanza. Split irradiazione minimizza la tossicità epatica acuta e quindi riduce il mouse letalità dovuta al protocollo irradiazione. </li><li> Dopo l&#39;irraggiamento, tenere i topi in trattamento con antibiotici per due settimane. Dare antibiotici con l&#39;acqua potabile a 2,5-5 mg di Baytril / kg. </li><li> Di 2 - 4 ore dopo la seconda irradiazione, topi trapiantati con cellule del midollo osseo del donatore per via endovenosa. Effettuare l&#39;iniezione di cellule del donatore via retro-orbitale sinusale iniezione 13 e sotto anestesia isoflurano utilizzando volume massimo di 100 ml di PBS contenente 2 x 10 6 cellule. </li><li> Quattro settimane dopo il trapianto, valutare attecchimento in un campione di 100ml di sangue periferico tramite citometria a flusso utilizzando una macchina in grado di leggere almeno due colori. Per distinguere tra donatore e cellule riceventi radio resistenti, utilizzare commercialmente disponibile CD45.1 fluorescente e anticorpi anti-topo CD45.2 utilizzando diluizioni tra 1/100 e 1/200. Attecchimento ottimale delle cellule del donatore deve essere almeno l&#39;80% del totale delle cellule ematopoietiche nel sangue periferico. </li></ol></li><li> La vaccinazione e mirata esaurimento <ol><li> Vaccinare i topi con il vaccino antinfluenzale vivo attenuato ricombinante via intranasale. Per la vaccinazione del mouse, anestetizzare i topi con il 5% isoflurano in ossigeno utilizzando un vaporizzatore di precisione. Utilizzare una pipetta 200 microlitri di somministrare 50 ml di PBS contenente 10 3 formanti placca unità (PFU) del vaccino. </li><li> Per generare il modello finale del mouse, trattare i topi con 50 ng di DT diluito in 50 ml di PBS intranasale. Somministrare DT goccia a goccia direttamente alle narici di topi anestetizzati come descritto in 3.3.1. Mi TreatCE con dosi giornaliere che iniziano il trattamento in giorno -2 prima della vaccinazione fino al giorno 2 post-vaccinazione (Figura 3B). </li></ol></li></ol>

<ol>
	<li>Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understand+memory,+design+better+vaccines.">Understand memory, design better vaccines.</a> Nat. Immunol. 12, 463-465 (2011).</li><li>Gaucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yellow+fever+vaccine+induces+integrated+multilineage+and+polyfunctional+immune+responses.">Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses.</a> J. Exp. Med. 205, 3119-3131 (2008).</li><li>Querec, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+biology+approach+predicts+immunogenicity+of+the+yellow+fever+vaccine+in+humans.">Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans.</a> Nat. Immunol. 10, 116-125 (2009).</li><li>Banchereau, J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+the+control+of+immunity.">Dendritic cells and the control of immunity.</a> Nature. 392, 245-252 (1998).</li><li>Mart&#237;nez-Sobrido, L., Garc&#237;a-Sastre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+influenza+virus+from+plasmid+DNA.">Generation of recombinant influenza virus from plasmid DNA.</a> J Vis Exp. 3, (2010).</li><li>Haniffa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Tissues+Contain+CD141hi+Cross-Presenting+Dendritic+Cells+with+Functional+Homology+to+Mouse+CD103++Nonlymphoid+Dendritic+Cells.">Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.</a> Immunity. 37, 60-73 (2012).</li><li>Haniffa, M., Collin, M., Ginhoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+functional+specialization+of+dendritic+cells+in+human+and+mouse.">Ontogeny and functional specialization of dendritic cells in human and mouse.</a> Adv. Immunol. 120, 1-49 (2013).</li><li>Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+depletion+of+CD11c++dendritic+cells+abrogates+priming+of+CD8++T+cells+by+exogenous+cell-associated+antigens.">In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.</a> Immunity. 17, 211-220 (2011).</li><li>Lahl, K., Sparwasser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+depletion+of+FoxP3++Tregs+using+the+DEREG+mouse+model.">In vivo depletion of FoxP3+ Tregs using the DEREG mouse model.</a> Methods Mol. Biol. 17, 211-220 (2011).</li><li>Duffield, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+depletion+of+macrophages+reveals+distinct,+opposing+roles+during+liver+injury+and+repair.">Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.</a> J. Clin. Invest. 115, 56-65 (2005).</li><li>Meredith, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+zinc+finger+transcription+factor+zDC.">Expression of the zinc finger transcription factor zDC.</a> J. Exp. Med. 209, 1153-1165 (2012).</li><li>Gir&#243;n, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+Polyinosinic-Polycytidylic+Acid+Improves+Protection+Elicited+by+Replicating+Influenza+Vaccines+via+Enhanced+Dendritic+Cell+Function+and+T+Cell+Immunity.">Mucosal Polyinosinic-Polycytidylic Acid Improves Protection Elicited by Replicating Influenza Vaccines via Enhanced Dendritic Cell Function and T Cell Immunity.</a> J. Immunol. 193, 1324-1332 (2014).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+animal+cells:+a+manual+of+basic+technique.">Culture of animal cells: a manual of basic technique.</a> bib.usb.ve. , (1983).</li><li>Cox, R. J., Brokstad, K. A., Ogra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus:+immunity+and+vaccination+strategies.+Comparison+of+the+immune+response+to+inactivated+and+live,+attenuated+influenza+vaccines.">Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines.</a> Scand. J. Immunol. 59, 1-15 (2004).</li><li>Gowdy, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+CD8(+)+T+cell+immunity+after+allogeneic+bone+marrow+transplantation+leads+to+persistent+and+severe+respiratory+viral+infection.">Impaired CD8(+) T cell immunity after allogeneic bone marrow transplantation leads to persistent and severe respiratory viral infection.</a> Transpl. Immunol. 32, 51-60 (2015).</li></ol>

The authors have nothing to disclose.

In questo studio viene descritto come la genetica e modelli di mouse chimerici inversa può essere utilizzata per chiarire i meccanismi fisiologici e molecolari dell&#39;immunità indotta dal vaccino. Influenza genetica inversa è stabilito in molti laboratori e ha svolto un ruolo principale nella comprensione patogenesi dell&#39;influenza, la replica, e la trasmissione 17. Un punto chiave nel nostro protocollo è il salvataggio di vaccini antinfluenzali adattate freddo esprimono epitopi stranieri. Mentre la ...

La generazione di memoria immunologica in assenza della malattia è la base fisiologica di vaccinazione efficace 1. Recentemente, sistemi di approcci basati biologia, hanno rivelato che i vaccini di successo come il vaccino contro la febbre gialla, inducono una forte induzione di risposte immunitarie innate e l&#39;attivazione di diversi sottoinsiemi di cellule dendritiche (DC), che a loro volta, portano a multilineare attivazione di antigene specifiche cellule T 2,3. Dal momento che le DC sono l&#39;unica popolazione di cellule immunitarie con la possibilità di attivare le cellule T naïve antigene-specifiche 4, lo studio della loro funzione durante la vaccinazione è fondamentale per capire le risposte immunitarie ai vaccini e per progettare strategie future contro gli agenti patogeni difficili. Un sistema che consente di risalire diverse sottopopolazioni PVS durante la risposta immunitaria ai vaccini sarebbe auspicabile, al fine di stabilire un accurato cinetica della migrazione DC a tessuti linfoidi, e quindi quello di fornirecomprensione dei meccanismi fisiologici responsabili per l&#39;avvio di specifici vaccini immunità adattativa. Genetica inversa a base di approcci offrono la possibilità di generare modificato, vaccini vivi attenuati che possono essere utilizzati in via sperimentale con questo scopo. Dal momento della sua attuazione in materia di ricerca di influenza, genetica inversa basata plasmide-è stato ampiamente impiegato per generare ceppi influenzali ricombinanti compreso LAIVs. Protocolli standard per salvare virus influenzali ricombinanti richiedono multi-trasfezione di linee altamente transfectable cellulari con plasmidi ambisense (che producono sia positivi che negativi senso RNA) che contengono i segmenti virali otto influenzali, nonché di amplificazione in un sistema permissiva come Madin-Darby rene canino ( MDCK) cellule e / o di gallina embrionate uova 5. Tuttavia, l&#39;applicazione di genetica inversa per generare strumenti molecolari per studiare i meccanismi immunitari della vaccinazione rimane inesplorato. La generazionedi nuovi modelli di mouse che permettono specifico esaurimento delle sottopopolazioni di cellule del sistema immunitario, tra cui DC, ha aperto nuove possibilità per comprendere i meccanismi immunitari fondamentali alla base di protezione del vaccino-suscitato. Il confronto tra le funzioni sottoinsieme DC nei topi e nell&#39;uomo ha rivelato che, in larga misura, mouse e DCS umani sono funzionalmente omologhe 6,7, questi risultati, suggeriscono fortemente che lo sviluppo di modelli murini consentendo specifica esaurimento delle DC nello stato stazionario e in condizioni infiammatorie, può servire per capire la fisiologia delle risposte DC nell&#39;uomo. Negli ultimi anni una serie di modelli di topo sono state generate portando transgeni esprimenti il recettore simian tossina difterica (DT) (DTR) sotto il controllo della regione del promotore di un gene di interesse 8,9. Dal momento che i tessuti di topo non naturalmente esprimono DTR, questi modelli consentono esaurimento condizionale di sottoinsiemi di cellule che trasportano il gene targeting di interesse sul inoculazione mouse con DT. Così, il nostro ability ad esaurire DC specifici e altri leucociti in vivo durante i processi fisiologici, è stata notevolmente rafforzata dallo sviluppo di ro-based DTR. Tuttavia, mentre questi modelli di topi transgenici sono stati ampiamente utilizzati per comprendere l&#39;ontogenesi del sistema immunitario, la loro applicazione per lo sviluppo del vaccino è stato appena testato. Qui, combinando l&#39;influenza genetica inversa e competitivi chimere del midollo osseo a base di DTR, proponiamo un metodo per studiare la cinetica di vaccino immunità così come la funzione del gene individuo durante la risposta immunitaria ai vaccini in vivo. L&#39;applicazione di questa tecnologia per la valutazione preclinica di nuovi vaccini contro le malattie infettive difficili potrebbe contribuire a razionalizzare la progettazione di vaccini e per testare vaccini candidati in vivo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium (DMEM 1X)</td>
			<td>Gibco RL-Life Technologies</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti MEM</td>
			<td>Gibco RL-Life Technologies</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen-Life Technologies</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10.000 U/ml)</td>
			<td>PAA</td>
			<td>p11-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryonated eggs</td>
			<td>Valo biomedia Gmbh</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1X)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;M Nylon Filters</td>
			<td>Greiner-Biorad</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing buffer (RBCL) 10X</td>
			<td>BD Bioscience</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16/CD32 Mouse BD Fc Block (2.4G2)</td>
			<td>BD Pharmigen</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Anti-mouse SIINFEKL-H2kb (25 D1.16)</td>
			<td>Biolegend</td>
			<td>141605</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Anti-mouse CD11c (HLA3)</td>
			<td>BD Biosciences</td>
			<td>553802</td>
			<td></td>
		</tr>
		<tr>
			<td>eFluor 450-Anti-mouse MHCII (Md/114.15.2)</td>
			<td>eBioscience</td>
			<td>48-5321-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Pe-Cy7-Anti-mouse CD11b (M1/70)</td>
			<td>Biolegend</td>
			<td>101216</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp/Cy5.5-Anti-mouse CD103 (2E7)</td>
			<td>Biolegend</td>
			<td>121416</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Anti-mouse CD45.1 (A20)</td>
			<td>eBioscience</td>
			<td>12-0453-82</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-Anti-mouse CD45.2 (1O4)</td>
			<td>BD Bioscience</td>
			<td>562130</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp-eFluor710 -Anti-mouse CD8a (53-6.7)</td>
			<td>eBioscience</td>
			<td>46-0081-80</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Cy7-Anti-mouse CD3&#949; (145-2611)</td>
			<td>Biolegend</td>
			<td>100325</td>
			<td></td>
		</tr>
		<tr>
			<td>eFluor450-Anti-mouse CD4 (GK 1.5)</td>
			<td>eBioscience</td>
			<td>48-0041-80</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE Proliferation dye</td>
			<td>eBioscience</td>
			<td>65-0850-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril 2.5%</td>
			<td>Bayer</td>
			<td>65-0850-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Dymethil-Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin&#160;</td>
			<td>Molecular probes&#160;</td>
			<td>O23020</td>
			<td></td>
		</tr>
		<tr>
			<td>Diphteria Toxin (DT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0564</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-TPCK</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACsCanto II Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo cell analysis software 9.5</td>
			<td>Flowjo inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>Life technologies</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Automatic Cell Counter</td>
			<td>Invitrogen-Life Technologies</td>
			<td>C10227</td>
			<td></td>
		</tr>
	</tbody>
</table>

intranasal administration of recombinant influenza vaccines in chimeric mouse models to study mucosal immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

Generazione di ricombinanti vaccini influenzali vivo attenuato può essere ottenuto mediante trasfezione di plasmidi che codificano le otto segmenti del virus dell&#39;influenza sotto il controllo di promotori bidirezionali 5. Un vaccino influenzale adattato fredda solito contiene sei segmenti di un ceppo adattato fredda, nonché l&#39;HA e NA del ceppo influenzale di scelta (ad esempio, H1N1) (Figura 1A). Il principio di freddo-adattamento si basa sulla replicazione virale limitato ...

Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

intranasal-administration-recombinant-influenza-vaccines-chimeric

Research

JoVE Journal

Immunology and Infection

12.4K Views.  Heinrich Pette Institute, Leibniz Institute for Experimental Virology. There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Video: Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Generazione di influenza da virus ricombinante del DNA plasmidi

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Ricerca

Immunologia e infezioni

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocollo per ricombinante RBD-based Vaccines SARS: Preparazione di proteine, vaccinazione degli animali e la rivelazione di neutralizzazione

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Analisi quantitativa di tutti influenza di tipo A Hemagglutinins e neuraminidasi virale utilizzando anticorpi Universale di saggi semplice Slot Blot

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

Esaminando il ruolo della rinofaringeo associata Tissue linforeticolare (NALT) nelle risposte mouse ai vaccini

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Un flusso di Adesione saggio per studiare Leucocyte reclutamento a epatici umani sinusoidali Endotelio in condizioni di stress Shear

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Intra-tracheale Amministrazione<em&gt; Haemophilus influenzae</em&gt; In modelli murini per studiare infiammazione delle vie aeree

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

L&#39;efficacia e la protezione polmonare risposta immunitaria seguito sottocutanea e intranasale BCG Amministrazione nei topi

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Misurare Influenza neuraminidasi Inibizione anticorpo I titoli da Enzyme-linked lectina Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Utilizzando modelli di Zebrafish di umana virus infezioni allo schermo farmaci antivirali e caratterizzare host immune cellulo Risposte

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...