1. La crescita di H. Capsulatum <ol><li> Preparare un armadio di biosicurezza per lavorare con il fungo pulendo l&#39;armadio con candeggina al 10% seguiti da 20 min irradiazione UV. H. capsulatum in fase di lievito richiede biosicurezza livello (BSL) pratiche II, mentre la forma dello stampo richiede BSLIII. Utilizza il form lievito nei seguenti passi. </li><li> Avanti preparare un tubo da 15 ml con 5 ml di PBS. Raccolta una colonia di H. capsulatum (ceppo ATCC Hc G217B) da una piastra di agar sangue BHI [glucosio 10 g / L, cisteina 0,1 g / L, penicillina-streptomicina 1% e pecore globuli rossi 50 ml / L (Colorado Serum Co., Colorado, USA) ] coltivati ​​a 37 ° C o prelevare un &#39;aliquota da stock lievito congelato a -80 ° C e aggiungere al PBS. Vortex le cellule vigorosamente e centrifugare per 1100 xg per 10 minuti a temperatura ambiente. Rimuovere con attenzione il sopranatante senza disturbare il pellet e aggiungere 10 ml di PBS fresco. Ripetete questa procedura per tre volte. Sospendere le cellule in 5 ml di PBS e il trasferimento in una provetta da 50 ml. </li><li> Per distruggere le cellule aggregate, passare la sospensione di cellule di lievito con un 26 G1 / 2 ago u cantare una siringa da 10 ml 5 volte in un armadio biosicurezza (con protezione del viso). Per isolare piccoli, lievito uniformemente dimensioni, centrifugare le cellule a 55 g per 1 minuto a temperatura ambiente e quindi rimuovere la parte superiore da 1 ml. </li><li > Aggiungere la sospensione cellulare ad una beuta sterile contenente 100 ml di terreno HAM F12 (GIBCO) integrata con 16 g / L di glucosio, 1g / L di acido glutammico, 8.4mg / L cistina, 6 g / L acido glutammico e poi crescere le cellule a 37 ° C per 48 ore in un incubatore con agitazione 150rpm. </li></ol> 2. Ibridomi crescita e di purificazione degli anticorpi monoclonali <ol><li> Trasferire il ibridomi selezionati e medie DMEM contenente 10% siero fetale bovino, il 10% NCTC, 1% di aminoacidi non essenziali e 1% di penicillina-streptomicina. Crescere le cellule in un pallone di colture cellulari (Becton Dickinson) per 5-7 giorni a 37 ° C / 5% di CO 2. </li><li> Centrifugare il supporto a 1100 xg per 10 minuti a temperatura ambiente e raccogliere tutte le sopranatante senza disturbare il pellet. </li><li> Successivamente, purificare il supernatante libero da cellule usando una proteina A / G di resina FPLC. </li><li> La concentrazione mAb può essere calcolato da una cattura ELISA (1) utilizzando un controllo isotipo IgG di serie (Figura 1B). </li></ol> 3. Histoplasma capsulatum Preparazione dell&#39;inoculo <ol><li> Prendete un h 48 Histoplasma capsulatum (ceppo ATCC Hc G217B) cultura fase lievito coltivato in HAM F-12 di media. </li><li> Centrifugare le cellule a 1100 xg per 10 min. Eliminare il supernatante e aggiungere PBS fresco. </li><li> Ripetere la procedura di 3,2 3 volte. </li><li> Passare la sospensione di cellule di lievito 5 volte attraverso un 26G1 / 2 aghi con una siringa. </li><li> Centrifugare la sospensione a 55 g per 1 minuto a gruppi di cellule pellet residuo. Trasferire il surnatante contenente la sospensione singola cella in una nuova provetta. </li><li> Enumerare la sospensione singola cella utilizzando un hematocytometer. </li><li> Regolare la concentrazione delle cellule al fine di raggiungere 1,25 x 10 7 lievito (sopravvivenza) o 5,0 x 10 6 (CFU, citochine e istologia) in un suspention di &lt;50 microlitri (Figura 2). </li></ol> 4. Somministrazione intraperitoneale del MAbs <ol><li> Sollevare il topo per la coda e la nuca del collo (Fig. 3A). Immobilizzare il mouse per la collottola del collo theears il più vicino possibile (assicurarsi di assumere abbastanza pelle in modo che il mouse cannotturn la testa a mordere la persona che gestisce esso; Figura 3B e 3C). </li><li> Stabilizzare il dito withthe piccola coda leggermente premuto contro il palmo della mano (Fig. 3D). </li><li> Tampone della zona di iniezione con etanolo al 70% prima di inserire l&#39;ago e per aspirare a cercare il sangue prima di iniettare. </li><li> Utilizzare il 26G1 / 2 a piercethe pelle e abdominalmuscles per iniettare la soluzione mAb contenente 500μg (PBS, il controllo degli anticorpi isotipo o il mAb da testare, diluito in 1 ml di PBS) nella sinistra thelower o il basso a destra dell&#39;addome (cavità intraperitoneale), con l&#39;animale nella posizione a testa in giù, avendo cura di evitare gli organi membrana interna andother (Figura 3E). </li><li> Attendere brevemente prima di ritirare il needleto ridurre la probabilità di perdite. </li><li> Attendere almeno due ore prima di procedere alla fase successiva. </li></ol> 5. Topo Anestesia e infezioni intranasale <ol><li> Preparare l&#39;anestesia con ketamina e xilazina a 100 mg / kg e 10 mg / kg, rispettivamente. Eseguire la somministrazione intraperitoneale di PBS, il controllo degli anticorpi isotipo, o anticorpi sperimentali, come descritto nel protocollo di accompagnamento scritto. Attendere due ore dopo l&#39;iniezione mAb prima anestetizzare il mouse e procedere con l&#39;infezione intranasale. Preparare ketamina anestesia utilizzando e xilazina a 100 mg / kg e 10 mg / Kg, rispettivamente (7).<html> </li><li> Durante il periodo di 2 ore di attesa, preparare l&#39;inoculo Histoplasma capsulum come descritto nel protocollo di accompagnamento scritto. </li><li> Procedere secondo il itens 5,1-5,5. Toe pizzicare gli animali con una pinzetta e di verificare l&#39;assenza di riflessi e confermare che l&#39;anestesia ha funzionato. Dopo anestesia, delicatamente sospendere il mouse per i suoi denti davanti ad una stringa di nylon o cotone. Utilizzare 2 stand colonna di legare le corde al fine di sospendere gli animali. </li><li> Lentamente amministrare circa 50 microlitri inoculo in un nare utilizzando una micropipetta, mentre segue con attenzione il tasso di respirazione del mouse. Lasciare che il mouse per riposare in questa posizione per 2-5 minuti dopo l&#39;infezione intranasale per facilitare la deposizione del lievito nei polmoni dell&#39;animale (Figura 3F). </li><li> Dopo il mouse è stato infettato, mantenere il mouse in un monitorata, ambiente caldo (temperatura tra i 25 ei 37 ° C), con cura utilizzando una lampada di calore, se necessario, fino a che non recupera dalla anestesia. Quando la sveglia mouse, tornare a una gabbia pulita con accesso ad libitum a cibo e acqua. Le gabbie vengono restituiti al nostro impianto di animali e tenuti in una stanza pulita animale (BLSI). </li></ol> 6. Studi di sopravvivenza <ol><li> Valutare gli animali infetti e mock-infettate clinicamente per tachipnea, letargia, ottundimento, e perdita di peso. Gli animali devono essere controllate due volte al giorno da parte dei membri di laboratorio e ogni giorno da custodi degli animali. </li><li> Con istoplasmosi murino, fino quasi alla fine della vita, ci sono di solito segni evidenti dell&#39;infezione diverso da un lieve aumento della frequenza respiratoria. Con malattia avanzata, il che avviene di solito da 10-11 giorni, i topi diventano significativamente tachypneic e hanno diminuita attività. Di solito sono rapidamente diventati moribondo e scadenza. </li><li> Gli animali in difficoltà dovrebbero essere opportunamente eutanasia con CO 2 in una camera dedicata, gli animali deceduti devono essere enumerati tutti i giorni. </li></ol> 7. CFU Studi, Istologia e citochine Studi <ol><li> Preparare provette da 15 ml con 10 ml PBS per ogni organo da raccogliere. </li><li> Eutanasia degli animali 7 e 14 giorni dopo l&#39;infezione con un inuculum subletale (5,0 x 10 6) come precedentemente indicato e rimuovere la, milza polmoni e fegato immediatamente. </li><li> Rimuovere un pezzo di organo da valutare ed effettuare la fissazione in formalina durante la notte. Le sezioni saranno esaminate al microscopio per la valutazione patologica. </li><li> Preparare 50 ml provette coniche con 5 ml di PBS superato con 70 filtri cellule micron per ogni organo deve essere omogeneizzato. Macerare ogni restante parte degli organi separatamente nel 50 ml provette sterili con pistoni siringa 5 ml. </li><li> Poi, in serie diluire omogenati d&#39;organo (1:100, 1:1000 e 1:5000) e 100μL piatto di ogni diluizione in BHI piastre di agar-sangue (2). </li><li> Aggiungi compresse inibitore della proteasi per il omogenati in base alle istruzioni del produttore (Roche, Germania). </li><li> Incubare le piastre a 37 ° C per un massimo di 14 giorni e CFU enumerare. </li><li> Centrifugare omogenati d&#39;organo a 4000xg per 10 minuti e rimuovere surnatanti organo, whish saranno utilizzati utilizzati nel test citochine effettuate secondo metodi standard secondo il produttore. </li></ol> 8. Anticorpo monoclonale di protezione contro H. Capsulatum dipende dalla specificità anticorpale e Isotipo <ol><li> MAb isotipo è fondamentale per la protezione contro H. capsulatum, come topi trattati con anticorpi monoclonali IgG1 o IgG2a di Hsp60 ha prolungato significativamente la sopravvivenza rispetto ai topi di controllo ricevere un mAb IgG2b di Hsp60 o sia un isotipo di controllo mAb o PBS (Figura 4). </li><li> Inoltre, in topi che avevano ricevuto anticorpi monoclonali di protezione, c&#39;è stata una riduzione significativa del numero di CFU polmonare e splenica al 7 ° giorno e diminuisce di 2 e 2,5 unità log di numeri lievito nei polmoni al giorno 14 (Figura 5). </li></ol><img src="/files/ftp_upload/2532/2532fig1.jpg" alt="Figura 1" /> Figura 1: Crescita di H. capsulatum e ibridomi. (A) HAM liquido preparazione F-12 la cultura da una singola colonia ottenuta da H. capsulatum coltivate su un BHI piastre di agar-sangue. (B) la crescita degli ibridomi in fiasche di coltura cellulare e purificazione mAb da proteina A / G cromatografia di affinità con resina. <img src="/files/ftp_upload/2532/2532fig2.jpg" alt="Figura 2" /> Figura 2: H. capsulatum preparazione inoculo di infezione intranasale. Sospensione di cellule ottenute dopo H. capsulatum interruzione aggregati da siringa e centrifugazione viene enumerato contando, in un emocitometro. La concentrazione delle cellule è regolata a 1,25 x 10 7 (esperimenti di sopravvivenza) o 5 x 10 6 (CFU, citochine e istologia) a &lt;50 microlitri. upload/2532/2532fig3.jpg &quot;alt =&quot; Figura 3 &quot;/&gt; Figura 3: la gestione del mouse durante mAb amministrazione. (A) Il mouse è preso per la coda e (B e C) immobilizzato tenendo la nuca del suo collo il più vicino possibile theears. (D) La coda è tenuta tra il mignolo e il palmo. (E) La soluzione mAb viene iniettato tramite un 26G1 / 2 ago. (F) Il H. capsulatum inoculo è somministrato per via intranasale pipettando gentilmente la sospensione cellulare in un Nare. <img src="/files/ftp_upload/2532/2532fig4.jpg" alt="Figura 4" /> Figura 4: MAbs di Hsp60 possono alterare la patogenesi di istoplasmosi. iniezioni ip con 500 mcg di IgG1, o IgG2a mAbs 2 ore prima di infezione prolungato significativamente la sopravvivenza (p &lt;0,05, rispetto ai controlli), ma un IgG2b mAb. <img src="/files/ftp_upload/2532/2532fig5.jpg" alt="Figura 5" /> Figura 5: CFU determinazioni nei polmoni a 7 e 14 giorni dopo una sfida subletale intranasale con 5x10 6 lieviti Hc ip di topi trattati con anticorpi monoclonali selezionati per Hsp60 o irrilevanti mAb ha mostrato una riduzione degli oneri fungine per IgG1 e IgG2a animali trattati con anticorpi monoclonali. Barre nere rappresentano CFU al giorno 7 e barre bianche 14 giorni dopo l&#39;infezione (* p &lt;0,001 a 7 giorni e ** p &lt;0,001 a 14 giorni dall&#39;infezione).

<ol>
	<li>Casadevall, A., Mukherjee, J., Scharff, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+based+ELISAs+for+cryptococcal+polysaccharide.">Monoclonal antibody based ELISAs for cryptococcal polysaccharide.</a> J. Immunol. Methods. 154, 27-35 (1992).</li><li>Guimaraes, A. J., Frases, S., Gomez, F. J., Zancope-Oliveira, R. M., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+heat+shock+protein+60+alter+the+pathogenesis+of+Histoplasma+capsulatum.+Infect+Immun+77.">Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of Histoplasma capsulatum. Infect Immun 77.</a> , 1357-1367 (2009).</li><li>Guimaraes, A. J., Hamilton, A. J., de M Guedes, H. L., Nosanchuk, J. D., Zancope-Oliveira, 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=Biological+function+and+molecular+mapping+of+M+antigen+in+yeast+phase+of+Histoplasma+capsulatum.">Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum.</a> PLoS One. 3, e3449-e3449 (2008).</li><li>Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+antibodies+and+endemic+dimorphic+fungi.">Protective antibodies and endemic dimorphic fungi.</a> Curr Mol Med. 5, 435-442 (2005).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Jr, D. e. e. p. e., &amp;amp, G. S., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J Clin Invest. 112, 1164-1175 (2003).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Deepe, G. S., , ., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J. Clin. Invest. 112, 1164-1175 (2003).</li><li>Smith, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+laboratory+animals+to+some+injectable+anaesthetics.">Responses of laboratory animals to some injectable anaesthetics.</a> Lab Anim. 27, 30-39 (1993).</li></ol>

Nessun conflitto di interessi dichiarati.

Il protocollo presentato qui dimostra che mAb di H. capsulatum può modificare il corso di sperimentale istoplasmosi murini. MAbs di antigeni di superficie di patogeni possono modificare le complesse dinamiche che si verificano durante l&#39;interazione tra un host e un agente patogeno. Questo studio stabilisce che mAbs mediare protezione in un modello murino di istoplasmosi letale quando iniettato per via intraperitoneale, e suggerisce proteine ​​candidate per lo sviluppo del vaccino, come H2B, antigene M ...

<table border="1"><tbody><tr><th> Nome del materiale </th><th> Azienda </th></tr><tr><td> Cappa di sicurezza biologica (BSL2) </td><td></td></tr><tr><td> Provette da 15 ml </td><td> Falcon, BD </td></tr><tr><td> HAM F-12 di media </td><td> Gibco </td></tr><tr><td> 37 ° C agitatore </td><td></td></tr><tr><td> Vortice </td><td></td></tr><tr><td> 50 ml provette coniche </td><td> Falcon, BD </td></tr><tr><td> 26G1 / 2 aghi </td><td> BD </td></tr><tr><td> Siringa da 10 ml </td><td> BD </td></tr><tr><td> 250 mL fiaschi </td><td></td></tr><tr><td> FPLC (Fast cromatografia liquida proteina) del sistema </td><td> GE Helthcare </td></tr><tr><td> Lettore ELISA </td><td> Biotek </td></tr><tr><td> Anestesia (ketamina e xilazina) </td><td></td></tr><tr><td> Colonna sta </td><td></td></tr><tr><td> Nylon string </td><td></td></tr><tr><td> Calore lampada </td><td></td></tr><tr><td> Filtri cellule 70μm </td><td> Falcon, BD </td></tr><tr><td> BHI piastre di agar </td><td> Gibco </td></tr></tbody></table>

passive administration of monoclonal antibodies against h. capsulatum and others fungal pathogens

Lo scopo l&#39;uso di questa metodologia è 1) per avanzare la nostra capacità di proteggere le persone con anticorpi o vaccini per prevenire o curare l&#39;istoplasmosi causata dal fungo Histoplasma capsulatum e 2) per esaminare il ruolo dei fattori di virulenza come bersaglio per la terapia. Per generare anticorpi monoclonali, i topi sono immunizzati, le risposte immunitarie sono valutati mediante un solido sistema di fase ELISA sviluppato nel nostro laboratorio, e la migliore topi risponditore vengono selezionati per l&#39;isolamento di splenociti per la fusione con cellule ibridomi. Topi C57BL / 6 sono stati ampiamente utilizzati per studiare H. patogenesi capsulatum e fornire il miglior modello per ottenere i dati richiesti. Al fine di valutare il ruolo del mAbs in infezione, i topi sono intraperitoneale somministrato sia con mAb a H. capsulatum o isotipo controllo di pari mAb e poi infettato da entrambi per via endovenosa (ev), intraperitoneale (ip), o intranasale (in) percorsi. Nella letteratura scientifica, l&#39;efficacia di anticorpi monoclonali per le infezioni fungine nei topi si basa sulla mortalità come un punto finale, in collaborazione con le unità Formin colonia (CFU) valutazioni in momenti precedenti. Sopravvivenza (tempo al decesso) studi sono necessari in quanto rappresentano al meglio la malattia umana. Quindi, l&#39;efficacia del nostro intervento non sarebbe adeguatamente essere stabilito senza curve di sopravvivenza. Questo vale anche per stabilire l&#39;efficacia del vaccino o test di mutanti per la virulenza. Con istoplasmosi, i topi vanno spesso ad essere energici per morto diverse ore. La capacità di un intervento, come la somministrazione di un mAb inizialmente può proteggere un animale dalla malattia, ma la malattia può ricaduta che non potrebbero essere realizzati in breve esperimenti CFU. Oltre a saggi di sopravvivenza e carica fungina, esaminiamo la risposta infiammatoria all&#39;infezione (istologia, reclutamento cellulare, le risposte citochine). Per la sopravvivenza / ora per esperimenti di morte, i topi sono infettati e controllati almeno due volte al giorno per i segni di morbilità. Per valutare la carica fungina, istopatologia, e le risposte delle citochine, i topi sono l&#39;eutanasia in vari momenti dopo l&#39;infezione. Gli esperimenti sugli animali vengono eseguiti secondo le linee guida dell&#39;Istituto per gli studi sugli animali dell&#39;Albert Einstein College of Medicine.

Watch this Scientific Journal Video about Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens at JoVE.com

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Topi C57BL / 6 sono stati utilizzati per studiare la patogenesi Hc e fornire il modello migliore. Stiamo esplorando i potenziali benefici dell&#39;immunità umorale contro questo fungo e ha generato anticorpi monoclonali diversi [di istone H2B e 60kDa Heat Shock Protein] che abbiamo testato per la loro efficacia protettiva dopo la somministrazione intraperitoneale.

Amministrazione passivo di anticorpi monoclonali contro H. capsulatum E altri patogeni fungini

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

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Immunology and Infection

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

11.8K Views.  Albert Einstein College of Medicine. Topi C57BL / 6 sono stati utilizzati per studiare la patogenesi Hc e fornire il modello migliore. Stiamo esplorando i potenziali benefici dell'immunità umorale contro questo fungo e ha generato anticorpi monoclonali diversi [di istone H2B e 60kDa Heat Shock Protein] che abbiamo testato per la loro efficacia protettiva dopo la somministrazione intraperitoneale.

Video: Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

L&#39;infezione di embrioni di zebrafish con batteri patogeni intracellulari

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

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Immunologia e infezioni

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Valutare l&#39;attività anti-fungina isolato Alveolar macrofagi dal Microscopia confocale

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Trasformazione Biolistic di una fluorescente Tagged Gene nel Opportunistic Fungal patogeni<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati B

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Generazione di Murine anticorpi monoclonali da ibridomi Tecnologia

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generazione di varianti di fuga di neutralizzare l'Influenza Virus anticorpi monoclonali

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generazione di anticorpi monoclonali umani discriminante da rari B antigene-specifiche cellule circolanti nel sangue

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

Generazione di anticorpi monoclonali contro prodotti naturali

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Anticorpli uccisione test per valutare le risposte immunologiche contro batteri patogeni

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to&#160;opsono-phagocytic killing assays. An advantage of the opsono-adherence&#160;assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.&#160;&#160;&#160;&#160;&#160;&#160;

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is an alternative method to the opsono-phagocytic killing assay to evaluate the opsonic functions of antibodies in vaccine development.

Test opsono-aderenza per valutare gli anticorpi funzionali nello sviluppo del vaccino contro bacillus anthracis e altri agenti patogeni incapsulati

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...