This research was possible through funding from the University of Neuch&#226;tel. We would like to thank all the students that helped in improving this technique, namely our colleague Kevin Thievent. We would also like to thank the members of the Thomas Lab for making their laboratory available. We would like to thank Janet Teeple for her help with mosquito rearing. We would also like to thanks Loyal Hall in the laboratory of Pr. Tom Baker for his help in the preparation of the micro capillary glass tubes.

1. soluzione salina per l&#39;iniezione e la dissezione <ol><li> Preparare la soluzione salina aggiungendo NaCl, KCl, e CaCl 2 per acqua distillata per ottenere 1,3 mM NaCl, 0,5 mM KCl, e 0,2 mM CaCl 2 a pH = 6,8. </li><li> Aggiungere 1 ml di 0,1% di metil soluzione verde a 99 ml della soluzione salina per colorare le perle trasparenti. Questo è il metile verde &quot;soluzione per l&#39;iniezione&quot; 0,001%. </li><li> Quindi, aggiungere 5 ml di 0,1% di metil soluzione verde a 45 ml di soluzione salina. Questa metil verde &quot;soluzione dissezione&quot; 0,01% è 10 volte più concentrato in verde metile per facilitare la colorazione delle perle e la loro osservazione durante la dissezione. NOTA: Filtro sterilizzare il buffer se necessario. </li></ol> 2. capillare Preparazione <ol><li> Tubi di vetro microcapillare Calore-pull per ottenere una finissima punta leggermente più grande delle piccole perle (φ = 40 micron) 42. </li><li> Aprire e unEGOLARE ogni capillare rompendo la punta con una pinzetta o segando la punta. </li></ol> 3. Mosquito allevamento <ol><li> Posteriore A. gambiae a 26 ± 1 ° C, 70 ± 5% di umidità relativa, e una luce 12:12 hr: ciclo scuro. Mantenere femmine adulte e dare loro l&#39;accesso a una soluzione zuccherina al 10%. </li></ol> 4. Le selezioni Bead con la capillare <ol><li> Versare 0,009 g di perle di carica negativa (40-120 micron di diametro) in una capsula di 5 centimetri di Petri e aggiungere 5 ml di soluzione iniettabile di 30 minuti in anticipo. NOTA: L&#39;obiettivo è quello di lasciare il colorante colore le perline per effettuare la selezione tallone per l&#39;iniezione più facile. Autoclave le perle se non sono disponibili in un barattolo sterile. </li><li> Utilizzando un capillare montato in un bulbo capillare pipetta, selezionare visivamente il più piccolo tallone in 0,1 ml di soluzione salina al microscopio binoculare. NOTA: quando inoculando varie perline contemporaneamente, selezionare tre perle da un volume totale di 0,1 ml.</li></ol> 5. Manipolazione Mosquito e inoculazione <ol><li> Utilizzando un aspiratore insetto, inserire ogni zanzara in un tubo da 50 ml. </li><li> Raffreddare ogni zanzara brevemente ponendo la provetta in ghiaccio tritato (2-5 min). </li><li> Inserire una zanzara sul lato destro al microscopio binoculare. </li><li> Sotto uno stereomicroscopio, inoculare la zanzara con il capillare saldamente forando il lato sinistro della cuticola cavità toracica e poi iniettato il liquido e il tallone. NOTA: Fare attenzione a non danneggiare i muscoli del volo tenendo il capillare come perpendicolarmente alla zanzara possibile. </li><li> Rimuovere il capillare. </li><li> Utilizzando pinze entomologia piuma, posizionare ogni femmina singolarmente in un bicchiere di plastica 180 ml coperta con zanzariere; dare loro accesso a una soluzione di zucchero 10%. NOTA: E &#39;necessaria cautela con la soluzione di zucchero, come la zanzara può rimanere bloccati sulle gocce di soluzione di zucchero. </li></ol> 6. MosquitoDissezione e Fotografia Bead <ol><li> Verificare che le zanzare sono vivi 24 ore dopo l&#39;inoculazione. Se è così, controllare la loro condizione e mantenere solo le zanzare che sono in grado di volare. </li><li> Congelare queste zanzare a -20 ° C. NOTA: Possono essere conservate per diversi giorni o mesi prima della dissezione, se necessario. </li><li> Rimuovere le ali delle zanzare utilizzando pinzette sotto un microscopio binoculare a 32X di ingrandimento. Fissare le ali su vetrini con nastro adesivo trasparente. Annotare il codice utilizzato per ogni singolo zanzara. </li><li> Mettere i vetrini in uno scanner e la scansione a 1.200 dpi. NOTA: Verificare che il codice è leggibile per ogni singolo zanzara. </li><li> Su un vetrino per microscopio e in soluzione dissezione, utilizzare pinze per separare il torace dalla testa e l&#39;addome. </li><li> Aprire il torace con una pinza per recuperare le perline. NOTA: Beads che non si muovono all&#39;addome, non sono in contatto tra di loro e la maggior parte di essi galleggiare liberamente in tegli Emolinfa. Fare attenzione a non rompere il tallone con le pinze, come una perla non melanized può essere difficile da trovare. Attendere da 1 a 5 minuti per perline per ottenere il colore. </li><li> Trasferire le perle in una goccia di soluzione di dissezione su un vetrino da microscopio in vetro, prima di scattare la foto. NOTA: Non c&#39;è bisogno di lavare le perline. </li><li> Utilizzando un microscopio dotato di una fotocamera, scattare una immagine digitale di ciascuna sfera in un ambiente illuminazione standard e l&#39;ingrandimento 400X. NOTA: Non modificare l&#39;illuminazione tra le diverse immagini per consentire loro confronto. </li></ol> 7. Analisi Immagine: Bead melanizzazione <ol><li> Aprire il software di analisi delle immagini. Fare clic su &quot;File&quot; e poi su &quot;Apri&quot; per trovare un&#39;immagine tallone. </li><li> Clicca su &quot;analizzare&quot;. Clicca su &quot;misure fissate.&quot; Barrare la casella &quot;valore medio grigia&quot; nella finestra &quot;misure fissate&quot;. </li><li> Fare clic sull&#39;icona dello strumento selezione cerchio. Selezionare il perimetro tallone; rimanere inlato del tallone e di evitare la alone luminoso intorno. </li><li> Fare clic sul menu &quot;analizzare&quot;. Clicca su &quot;misura&quot;. Ottenere il valore di grigio medio e le dimensioni della sezione trasversale del cordone. <ol><li> Per ottenere il valore melanizzazione di ciascuna sfera, eseguire la seguente operazione utilizzando una calcolatrice o un calcolatore software: &quot;256 - valore medio grigio.&quot; La misura melanizzazione è un valore compreso tra 0 e 256, con 0 che rappresenta un&#39;immagine completamente bianca. </li></ol></li></ol>

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J., George, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Manipulation&amp;#34;+without+the+parasite:+altered+feeding+behaviour+of+mosquitoes+is+not+dependent+on+infection+with+malaria+parasites.">34;Manipulation&amp;#34; without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites.</a> P Roy Soc B-Biol Sci. 280 (1763), 20130711 (2013).</li><li>Voordouw, M. J., Lambrechts, L., Koella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+maternal+effects+after+stimulation+of+the+melanization+response+in+the+yellow+fever+mosquito+Aedes+aegypti.">No maternal effects after stimulation of the melanization response in the yellow fever mosquito Aedes aegypti.</a> Oikos. 117 (8), 1269-1279 (2008).</li><li>Paskewitz, S., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Plasmodium+refractory+and+susceptible+strains+of+Anopheles+gambiae+to+inoculated+Sephadex+beads.">Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads.</a> Dev comp immunol. 18 (5), 369-375 (1994).</li></ol>

The authors declare that they have no competing financial interests.

Questa tecnica di iniezione è utile per stimolare e studiare la risposta melanizzazione in zanzare. Ad esempio, qui abbiamo studiato l&#39;effetto del carico stimoli immunitario. Il passo fondamentale in questa procedura è per inoculare correttamente la zanzara. Qualsiasi danni eccessivi ai muscoli del volo o per la zanzara si può impedire la zanzara da alimentazione o può uccidere prima della dissezione. Un secondo passo fondamentale è quello di mantenere le zanzare sul ghiaccio abbastanza a lungo da metterli fuori senza ucciderli. Un piccolo contenitore faciliterà e accelerare il processo, in quanto consente un più stretto contatto con il ghiaccio. Infine, è necessaria particolare attenzione durante la dissezione, come pinzette possono altrimenti rompere le perline. Perline incorporati nel tessuto zanzara può richiedere più tempo per essere colorati dalla soluzione dissezione. L&#39;utilizzo di più tipi di perle (come neutrale, positivamente o negativamente carica perline o perle di vetro) porterà a risultati diversi, come may non tutti essere melanized allo stesso grado 27,43. Infatti, a fronte di perline carica neutra, perline carichi negativamente portano ad una maggiore variabilità nella risposta melanizzazione tra le zanzare 27. Perle di vetro, d&#39;altro canto, sono inerti nelle zanzare e non attivano la risposta melanizzazione affatto 27. perline carica negativa possono quindi essere preferito, ma il confronto tra le diverse sfere possono anche essere pertinente. Tuttavia, a seconda del tipo di tallone, un colorante diverso può essere richiesto per le soluzioni di iniezione e sezionamento; per esempio, colorante violetto cresolo dovrebbe essere usato al posto del verde metile quando si utilizza perline di vetro 27. Questa tecnica di iniezione è invasiva per la zanzara. Anche se i danni e lesioni possono essere ridotti al minimo e controllati, gli strumenti alternativi possono essere una scelta migliore, a seconda del disegno sperimentale. È difficile stimolare e misurare simultaneamente diverse risposte immunitarie nella stessa zanzara. Lavoroing sulle famiglie o gruppi di zanzare è necessario quando guardando le correlazioni tra le risposte immunitarie 28. Inoltre, perline sono solo parzialmente simile alla effettiva presenza di parassiti in un insetto. Innanzitutto, la risposta immunitaria è stimolata, e quindi la risposta fenotipica risultante, l&#39;incapsulamento, viene misurato, ma ciò che accade in mezzo è sconosciuta. Accoppiamento iniezioni con strumenti fisiologici o molecolari 10 - 12 in grado di migliorare la nostra comprensione di questa risposta melanizzazione. Per studiare i meccanismi alla base della risposta melanizzazione, l&#39;espressione di enzimi chiave o molecole prodotte durante la cascata pro-phenoloxidase può essere misurata 10 - 12. Tuttavia, concentrandosi esclusivamente sulla up- o down-regulation di tali molecole, senza conoscere le conseguenze reali della incapsulamento finale, può portare ad una valutazione inesatta del processo melanizzazione. Pertanto, lo studio ENCApsulation ed i meccanismi molecolari insieme sarebbe altamente informativo, nonostante il fatto che sono ciascuna una tecnica scientifica valida usati separatamente. Per concludere, questa è una tecnica per studiare l&#39;immunità e l&#39;evoluzione dell&#39;immunità. Vi è, ad esempio, una crescente evidenza di un legame tra resistenza agli insetticidi e suscettibilità zanzara parassiti della malaria. Questa tecnica di iniezione potrebbe essere utilizzata in un tale contesto, così come nelle prove sul campo. Una seconda direzione di lavoro potrebbe essere quella di combinare la stimolazione immunitaria con atterramenti gene per studiare percorsi immunitario in dettaglio.

Gli insetti si basano su risposte immunitarie per proteggersi contro i parassiti e patogeni 1 - 3 che violano attraverso la loro cuticola o il loro midgut dell&#39;epitelio 4. In zanzare, queste risposte sono efficaci contro i batteri, virus 5 6, nematodi filarial 7, e parassiti della malaria 1,8,9. In zanzare, una risposta immunitaria chiave è l&#39;incapsulamento di corpi estranei con la melanina 10 - 12. Questo incapsulamento può accadere nel midgut o in emolinfa sistema di circolazione 10 - 12. Questa risposta melanizzazione è il risultato del pro-phenoloxidase cascata 10 - 12, e può portare alla morte dei parassiti o alla loro fagocitosi. In zanzare adulte, in cui viene limitato il numero di cellule ematociti concentrati, melanizzazione è una risposta umorale, come contro i parassiti Plasmodium o nematodi filarial 7.In alcuni altri insetti, è direttamente le cellule hemocytes che si riuniscono intorno al parassita di melanize loro 7. Inoltre, la melanina è essenziale anche per molti altri processi fisiologici come la produzione di uova e ferite cuticola guarigione 7. La stimolazione della risposta immunitaria è utilizzato come strumento per studiare l&#39;immunità insetto in diversi sistemi modello sanità pubblica agricola e 13 - 18. E &#39;utilizzato in Anopheles gambiae zanzare, il principale vettore della malaria in Africa, per studiare le interazioni ospite-parassita 14 - 16,19. Queste tecniche si basano sulla capacità di insetti per individuare parassiti con i loro recettori pattern recognition (PRR) 2. Le zanzare possono anche rilevare altre molecole che interferiscono con la loro biologia come modelli molecolari associati ai patogeni (PAMPs), o rilevare le proprie cellule danneggiate a causa del rilascio di collagene ed acidi nucleici. La cellula immunitaria della zanzaras quali gli emociti sono utilizzati per la rilevazione 20 - 23. Le principali vie di segnalazione immunitario sono Imd, Toll, JAK / STAT 24, e l&#39;interferenza acido ribonucleico (RNAi) 25,26. Sia Toll e percorsi IMD influenzano la risposta melanizzazione e interagiscono con il pro-phenoloxidase cascata 10 - 12. Lo strumento standard utilizzato per stimolare la risposta melanizzazione è l&#39;inoculazione di una zanzara con un piccolo tallone nel emolinfa della cavità toracica. Il grado di melanina incapsulamento può quindi essere misurato 19 dopo aver ottenuto il tallone attraverso la dissezione della zanzara. Nella maggior parte degli studi, solo una perlina stato iniettato per zanzare 15,16,27, ma iniettando più perline è possibile al fine di studiare i limiti della risposta melanizzazione 19. Queste perle sono iniettati con una soluzione iniettabile (fisiologica) per ridurre i disturbi della fisiologia e zanzarel&#39;essiccazione della zanzara 15,16,27. Un colorante viene aggiunto a questa soluzione per facilitare la selezione tallone. È lo stesso per la soluzione dissezione utilizzato per recuperare il 15,16,27 tallone. Il vantaggio di inoculare insetti con stimoli non patogeno è la capacità di mettere a fuoco l&#39;effetto diretto sulla risposta immunitaria. Non ci sono effetti di complicazione a causa di parassiti patogenicità 28, immunosoppressione 29 - 31, o l&#39;evasione immune 31 - 34. Inoltre, le conseguenze delle stimolazioni su altri tratti della storia di vita, come la longevità o la fecondità, possono anche essere studiati. Così, i ricercatori che studiano l&#39;ecologia evolutiva possono richiedere tali strumenti 2,35,36. Ad esempio, i bombi immuno-sfidato hanno una durata di vita ridotta sotto fame. effetti negativi simili di stimolazione del sistema immunitario e le implementazioni sono state osservate in diversi modelli di invertebrati, spesso con conseguente breveer la durata della vita o il successo riproduttivo meno 13,27,37. Tali studi possono essere condotti in diversi ambienti 2,4,38. Stimolare l&#39;immunità è anche di interesse per coloro concentrarsi direttamente su immunopatologia 39,40. Questo protocollo si basa sulla inoculazione di perline con zanzare per stimolare la risposta melanizzazione e direttamente misurare la quantità di melanina. In questo modo lo studio quantitativo e qualitativo della risposta melanizzazione in contesti diversi sperimentali. Tale strumento può essere esteso alla stimolazione di altre risposte immunitarie, come la risposta antibatterico per batteri 41 termicamente uccisi. Può anche essere condotta in molti ambienti ecologici.

<table border="0" cellpadding="0" cellspacing="0" width="718">
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		<tr height="85">
			<td height="85" style="height:85px;width:301px;">Microcapillary glass tubes GB120TF-10</td>
			<td style="width:177px;">science-products.com</td>
			<td style="width:84px;">GB120TF-10</td>
			<td style="width:156px;">http://www.science-products.com/Products/CatalogG/Glass/Glass.html</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:301px;">Microcaps Capillary pipette bulb</td>
			<td style="width:177px;">Drumond</td>
			<td style="width:84px;">1-000-9000</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:301px;">negatively charged Sephadex CM C-25 beads</td>
			<td style="width:177px;">Sigma-Aldrich, Steinheim, Germany</td>
			<td style="width:84px;">C25120 SIGMA</td>
			<td style="width:156px;">need few to start</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:301px;">Methyl green</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:84px;">323829 ALDRICH</td>
			<td style="width:156px;">need few to start</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:301px;">Software ImageJ</td>
			<td style="width:177px;">opensource</td>
			<td style="width:84px;"></td>
			<td style="width:156px;">Version 1.47f7 or later</td>
		</tr>
	</tbody>
</table>

inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

La stimolazione della risposta immunitaria è un attrezzo comune negli studi invertebrati per esaminare l&#39;efficacia ei meccanismi di immunità. Questa stimolazione è basato sull&#39;iniezione di particelle non patogeni in insetti, come le particelle vengono rilevati dal sistema immunitario e indurrà la produzione di effettori immunitari. Ci concentriamo qui sulla stimolazione della risposta melanizzazione nella zanzara Anopheles gambiae. I risultati di risposta melanizzazione nel incapsulamento di particelle estranee e parassiti con uno strato scuro di melanina. Per stimolare questa risposta, le zanzare vengono inoculati con perline all&#39;interno della cassa toracica utilizzando tubi di vetro microcapillari. Poi, dopo 24 ore, le zanzare sono sezionati per recuperare le perline. Il grado di melanizzazione del cordone viene misurata utilizzando il software di analisi delle immagini. Beads non hanno gli effetti patogeni di parassiti, o la loro capacità di eludere o sopprimere la risposta immunitaria. Queste iniezioni sono un modo per misure efficacia immunitario e l&#39;impatto della stimolazione del sistema immunitario su altri tratti della storia di vita, come ad esempio la fecondità e la longevità. Non è esattamente lo stesso di studiare direttamente le interazioni ospite-parassita, ma è uno strumento interessante per studiare l&#39;immunità e la sua ecologia evolutiva.

<html> Zanzare non tutti hanno melanize le sfere nello stesso modo, come alcuni perle sono state coperte con meno melanina di altri (Figura 1). Infatti, alcune perle rimaste blu a causa di una mancanza di melanizzazione, mentre altri sono stati completamente scuro (Figura 1). Il valore melanizzazione stato standardizzato mediante interpolazione lineare per un valore compreso tra 0 (che corrisponde ad un cordone blu e unmelanized) e 100 (corrispondente ad una perlina scuro e fortemente melanized) (Figura 2a e 2b). Questi valori indicano il grado di melanizzazione delle perle e quindi, la forza della risposta immunitaria. Possiamo confrontare perline gli uni agli altri, confrontando i loro valori melanizzazione. La risposta melanizzazione generalmente segue una distribuzione gaussiana, che consente analisi statistiche classici come ANOVA o glm gaussiana. &quot;Fo: together.within-page keep-=&quot; 1 &quot;&gt; Abbiamo studiato la variazione della risposta melanizzazione rispetto al numero di perline contemporaneamente inoculate in una zanzara L&#39;obiettivo è stato quello di ottenere una migliore comprensione della variabilità e limiti. la risposta melanizzazione. la dimensione del tallone non ha avuto alcun effetto. Il valore medio tallone melanizzazione diminuito 71-50 quando il numero di perline inoculato aumentato (F IGURA 2). Ci sono alcuni limiti della risposta melanizzazione in una zanzara quando la sfida immunitario aumenta. Inoltre, la risposta melanizzazione diminuita per il tallone meno melanized in ogni zanzara riguardava il 71 al 35 in uno a tre branelli rispettivamente. Tuttavia, il valore melanizzazione del tallone più fortemente melanized rimasto sulla costante. La variabilità di melanizzazione aumenta con il numero di sfere. Lo sforzo melanizzazione non è stato uniforme tra tutte le perle in un dato zanzara,come un tallone è stata la priorità sugli altri. Questa tecnica di iniezione è utile per studiare la variabilità delle risposte immunitarie in zanzare. <img alt="Figura 1" src="/files/ftp_upload/55013/55013fig1.jpg" /> Figura 1: esempi immagine di un non-melanized, una parte melanized, e una perlina altamente melanized dopo la dissezione. A sinistra, centro e destra sono una perlina non melanized, un cordone parzialmente melanized, e una perlina altamente melanized, rispettivamente. <img alt="figura 2" src="/files/ftp_upload/55013/55013fig2.jpg" /> Figura 2: melanizzazione branello in funzione del numero di perline inoculate. (A) Ogni punto mostra la melanizzazione di un unico tallone (con valori che vanno da 0 melanizzazione per perline non melanized a 100 per quelli pesantemente melanized) e ogni colonna di punti rappresentano una zanzara individuale. ogni Mosquito è stato inoculato con uno, due, o tre perle. I punti solidi rappresentano il valore più alto melanizzazione per le zanzare, i punti incrociate rappresentano il valore melanizzazione intermedio, ei punti vuoti rappresentano il valore più basso melanizzazione. Le linee continue rappresentano il melanizzazione trattamento medio a tallone, le linee tratteggiate mostrano la media più alta melanizzazione, e le linee tratteggiate indicano la media della melanizzazione basso. (B) Media e errore standard della melanizzazione, la più alta melanizzazione e il melanizzazione più basso per il trattamento di inoculazione tallone. Il nero rappresenta il tallone melanizzazione medio in funzione del numero di sfere (con valori che vanno da 0 melanizzazione per perline non melanized a 100 per quelli pesantemente melanized). Il punto rosso rappresenta la media del più alto melanizzazione. Il triangolo blu rappresenta la media della melanizzazione più basso. I bar sono gli errori standard. Modificato da riferimento 19. &lt;/ P&gt;

Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

inoculando<em&gt; Anopheles gambiae</em&gt; Le zanzare con perline di indurre e misurare la risposta immunitaria melanizzazione

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

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Immunology and Infection

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

7.9K Views.  Université de Neuchâtel. Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

Video: Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Misurazione della carica batterica e Risposte immunitarie in topi infettati con Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

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Immunologia e infezioni

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

An Introduction to vespe parassite di<em&gt; Drosophila</em&gt; E la risposta immunitaria antiparassitari

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imaging intravitale del Timo mouse utilizzando 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Misurazione quantitativa della risposta immunitaria e nel sonno<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Quantificazione della risposta Burst respiratoria come un indicatore di Innate Immune Salute in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Usando RNA-interferenza per Indagare sulla risposta immunitaria innata in mouse macrofagi

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

L&#39;utilizzo di orofaringea intratracheale PAMP Amministrazione e lavaggio broncoalveolare per valutare la risposta immunitaria nei topi Host

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Due-fotone videomicroscopia Imaging dei leucociti durante la risposta immunitaria nel fegato Lipopolysaccharide-trattati del topo

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Un modello di topo per valutare risposta immunitaria innata per infezione da Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...