Ringraziamo Peter Staeheli per la fornitura di topi B6.A2G-MX1 e semre Özcelik consulenza tecnica. Questo lavoro è stato sostenuto dalla Fondazione per la Ricerca tedesco attraverso SFB829 e KN536 / 16, e la Colonia Fortune Programma / Facoltà di Medicina e Chirurgia, Università di Colonia.

Dichiarazione Etica. La preparazione di fogli epidermici di animali sacrificati viene effettuata in stretta conformità con le raccomandazioni della Guida di Landesamt für Natur, Umwelt e Verbraucherschutz, Nord Reno-Westfalia (Germania). Lo studio è stato approvato dal LANUV NRW (Numero 8.84-02.05.20.13.018). 1. Preparazione degli strumenti e cultura dei media <ol><li> Coltivare fogli epidermica in mezzo modificato Eagle Dulbecco (DMEM) / alto glucosio contenente glutamina dipeptide, il 10% FCS, 100 UI / ml di penicillina, e 100 mg / ml di streptomicina (di seguito denominato &quot;DMEM&quot;). </li><li> Per la preparazione di fogli epidermiche, sciogliere accuratamente dispasi II polvere (5 mg / ml) in PBS riscaldata (fino a 37 ° C). Filtrare la soluzione attraverso un filtro di 0,22 micron e utilizzarlo direttamente per il trattamento. </li><li> Per la preparazione di epidermica intero monta 13, preparare il blocco appassionatoer con 0,5% di latte in polvere, 0,25% di gelatina dall&#39;acqua fredda pelle di pesce e 0,5% Triton X-100 in TBS. Consentire sostanze per sciogliere incubando la miscela per almeno 2 ore a RT su un agitatore rotante. Anche preparare 0,2% Tween 20 in PBS come tampone di lavaggio. Per la fissazione, preparare 3,4% e 4% formaldeide in PBS. </li><li> Per la immunocolorazione di criosezioni, preparare un tampone bloccante con 5% di siero di capra normale e 0,2% Tween 20 in PBS. Per la fissazione, preparare 0,5% di formaldeide in PBS. </li></ol> 2. Preparazione di fogli epidermico da pelle murino <ol><li> Preparazione di epidermide da pelle posteriore neonato per gli studi di infezione <ol><li> Collocare un mouse decapitati (da 1 a 3 giorni dopo la nascita) in un piatto dissezione e tagliare le estremità e la coda vicino al torso per consentire la rimozione efficiente della pelle dal torso completa. Durante la rimozione delle estremità, cercare di mantenere i tagli più piccolo possibile di diametro. </li><li> Fissare il torso curvo con una pinza sottilend spostare delicatamente le forbici punta smussa sotto la pelle dal cranio caudale a lungo la parte posteriore per separare la pelle dal busto. Incidete la pelle lungo la parte posteriore. </li><li> Per l&#39;analisi FACS, isolamento di cheratinociti o la preparazione di RNA, staccare la pelle completo dal tronco con pinze per fissare il tessuto e le forbici punta smussata per spingere fuori il busto. Per la preparazione di criosezioni o supporti interi prendere solo pezzi di pelle indietro. </li><li> Tritate la pelle torna con un bisturi in 1-2 pezzi di circa 10 x 10 mm. Mettete i pezzi in un pozzetto di un 6-pozzetti con ~ 1 ml di PBS sterile. Assicurarsi che il lato dermico affaccia sul fondo. </li><li> Sostituire PBS da 1,5 ml dispasi II (5 mg / ml PBS) e incubare per entrambi 30 minuti a 37 ° C (per i supporti interi) o O / N a 4 ° C. Lavare 3 volte con PBS. Rimuovere delicatamente l&#39;epidermide come un foglio intatto utilizzando una pinza per riparare il derma sottostante e le altre pinze per sollevare l&#39;epidermide. Utilizzare un binocolo a fare questo passo easa. Trasferire il foglio epidermica in DMEM con il lato basale verso il fondo. Utilizzare immediatamente le lenzuola per gli esperimenti di infezione. NOTA: Utilizzare la preparazione di epidermide dalla pelle appena nato principalmente per l&#39;analisi FACS, isolamento di cheratinociti o la preparazione di RNA. A causa della sua fragilità, la preparazione di fogli infettati per criosezioni o supporti interi è meno adatto. </li></ol></li><li> Preparazione di epidermide da pelle coda adulto per gli studi di infezione <ol><li> Euthanize i topi all&#39;età di 1 a 3 mesi per dislocazione cervicale. Prendere coda invece di pelle indietro da allora i follicoli dei capelli lunghi integrati sul retro ostacolano separazione dei fogli epidermico intatte. Tagliare la coda di topi sacrificati e fessura nel senso della lunghezza con un bisturi. </li><li> Staccare la pelle dalle ossa e tritarlo con un bisturi in pezzi di circa 5 x 5 mm. Mettere fino a 4 pezzi in un unico bene e procedere con l&#39;incubazione in dispasi II come descritto nella sezione 2.1.5 NOTA: Utilizzare tai adultil pelle per preparare criosezioni o supporti interi. In alternativa l&#39;uso immediato, code possono essere immagazzinati o trasportati per circa 24 ore a 4 ° C senza perdere significativo l&#39;efficienza dell&#39;infezione. Se esce croce sono mantenuti fino a 48 ore, lenzuola epidermici potrebbero essere difficilmente infettati con HSV-1. </li></ol></li><li> Dissociazione dell&#39;epidermide per analisi FACS NOTA: Il rilevamento di superficie espressione della proteina mediante analisi FACS richiede adeguate tecniche di dissociazione cellulare che non danneggiano la superficie della cellula e la proteina di interesse. <ol><li> Incubare fogli epidermica appena nati in un 6-pozzetti con la parte basale a 1,5 ml / soluzione dissociazione cella ben ricombinante basato tripsina-per 15 minuti a RT e per 15 minuti a 37 ° C. </li><li> Fermare l&#39;incubazione con l&#39;aggiunta di 3 ml di DMEM. Dissociarsi cheratinociti utilizzando un curva pinza sottile per muovere delicatamente l&#39;epidermide in 6 pozzetti in modo che i cheratinociti si dissolvono. Raccogliere la sospensione cellulare e ripetere l&#39;operazione 3 voltesempre utilizzando DMEM fresco. NOTA: Questa procedura di dissociazione è opportuno rilevare nectin-1 sulla superficie dei cheratinociti. </li><li> In alternativa, incubare le lenzuola epidermiche di soluzione dissociazione cella priva di enzima (CDS) per 15 minuti a temperatura ambiente e 15 minuti a 37 ° C. Incubare altri 15 minuti a temperatura ambiente per irrigare delicatamente i cheratinociti pipettando CDS su e giù. </li><li> Raccogliere la sospensione cellulare e ripetere il passaggio di risciacquo 3 volte utilizzando DMEM fresco. NOTA: Questa dissociazione è atto a rilevare l&#39;espressione superficie HVEM. Lo svantaggio della soluzione CDS è che meno cellule sono dissociate che con soluzione ricombinante dissociazione cellulare basato tripsina. Oltre alla superficie espressione, espressione nucleare o citoplasmatica come espressione ICP0 può essere analizzato mediante FACS dopo dissociazione dei fogli epidermica infetti con soluzione ricombinante dissociazione cellulare basato tripsina. </li></ol></li><li> La dissociazione di epidermide per isolare cheratinociti o extratto RNA <ol><li> Mettere fogli epidermiche neonato in una piastra da 6 pozzetti con il lato basale verso il fondo del piatto che contiene 1,5 ml / soluzione ben ricombinante dissociazione cellulare basato tripsina. Incubare per 30 minuti a temperatura ambiente. Aggiungere 3 ml di DMEM e dissociare cheratinociti utilizzando un curva pinza sottile per spostare delicatamente l&#39;epidermide nel piatto. </li><li> Raccogliere la sospensione cellulare e ripetere l&#39;operazione 3 volte utilizzando DMEM fresco. Utilizzare questa sospensione cellulare per estrarre l&#39;RNA o alla cultura primari cheratinociti murini. </li></ol></li></ol> 3. L&#39;infezione di fogli epidermico con HSV-1 <ol><li> Preparare una soluzione di HSV-1 wt ceppo 17 11 in non meno di 500 microlitri DMEM e sostituire il mezzo dalla soluzione virus cui i fogli epidermici sono flottanti. Questo punto di tempo viene definito come tempo 0 12. Eseguire infezione di un foglio epidermica in un pozzetto di una piastra da 24 pozzetti a 37 ° C. Il calcolo del titolo virale è basato sul numero stimatodelle cellule nello strato basale per foglio epidermica (circa 2 x 10 5 cellule per foglio 5 x 5 mm). </li><li> Infettare con HSV-1 a circa 100 unità formanti placca (PFU) / cellulare e sostituire il medio virus contenente in DMEM a 1 ora pi NOTA: Nel caso di fogli epidermica dalla pelle neonato, tenere a mente che i fogli cominciano a dissociarsi durante l&#39;incubazione. </li></ol> 4. Visualizzazione delle cellule infettate <ol><li> Epidermal intero Monti 13 <ol><li> Fissare fogli epidermico con il 3,4% di formaldeide o per 2 ore a temperatura ambiente o O / N a 4 ° C. Lavare due volte con PBS e bloccare con 0,5% di latte in polvere, 0,25% di gelatina dall&#39;acqua fredda pelle di pesce e 0,5% Triton X-100 in TBS per 1 ora a RT 14. </li><li> Incubare le lenzuola O / N con topo anti-ICP0 (anticorpo monoclonale 11060) 15 diluito 1:60 in tampone di bloccaggio a temperatura ambiente. Per visualizzare la rete di filamenti intermedi incubare contemporaneamente policlonale di coniglio anti-Mouse cheratina 14 (100 ng / ml). </li><li> Lavare 4 a 5 volte con PBS-Tween 20 0,2% durante 4 ore a temperatura ambiente. Incubare O / N con AF488-coniugato anti-IgG di topo (1 mg / ml), IgG anti-coniglio AF555-coniugato (1 mg / ml), e DAPI (4 &#39;, 6-diamidino-2-fenilindolo) (100 ng / ml) in tampone di bloccaggio a RT. </li><li> Lavare con PBS-Tween 20 0,2% per 4 ore a temperatura ambiente. Fogli epidermico Mount con la loro parte basale sulla cima di un vetrino, incorporare nel mezzo di montaggio fluorescente e coprire con coprioggetto. </li></ol></li><li> Epidermal criosezioni <ol><li> Fogli epidermiche di età sono stati fissati in formaldeide al 4% in PBS per 20 minuti a RT e lavate 2 volte con PBS. Fogli fissi Incorpora in tessuto di congelamento medio e congelare in azoto liquido. Tagliare 8-10 sezioni micron con un criomicrotomo. </li><li> Fissare nuovamente le sezioni con 0,5% di formaldeide in PBS per 10 minuti a RT, lavare 2 volte con PBS, blocco con 5% di siero di capra normale e 0,2% Tween 20 in PBS per 30 minuti a RT, e quindi macchiare per 60 min con mOuse anti-ICP0 (anticorpo monoclonale 11060) 15 diluito 1:60 in tampone bloccante, seguito da 3 tempi di lavaggio con il tampone di bloccaggio. </li><li> Poi incubare con AF488-coniugato anti-IgG di topo (1 mg / ml) e DAPI (100 ng / ml) in tampone bloccante per 45 minuti a RT e lavare 3 volte. Sezioni incorporare nel mezzo di montaggio fluorescente e coprire con coprioggetto. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Quando i fogli epidermica di pelle adulta da C57BL / 6 sono infettati con HSV-1 a circa 100 PFU / cellula, osserviamo l&#39;infezione in quasi tutte le cellule dello strato basale dell&#39;epidermide interfollicolari mentre dosi virali inferiori correlano con le cellule meno infette e una più lenta progresso dell&#39;infezione. In generale, i follicoli piliferi mostrano un numero variabile di cellule infettate; mentre la maggior parte dei piccoli piuttosto cheratinociti che rivestono i follicoli dei capelli in via di s...

Herpes simplex virus (HSV) può causare una serie di malattie negli esseri umani da lievi lesioni mucocutanee semplici alle infezioni pericolose per la vita. HSV di tipo 1 (HSV-1) è prevalentemente associata a infezioni orofacciale e l&#39;encefalite, mentre HSV di tipo 2 (HSV-2) più probabile causa di infezioni genitali 1. Mentre non vi è un progresso significativo nella comprensione di come HSV entra nelle cellule in coltura, avvia l&#39;infezione e produce progenie virale, sappiamo poco del percorso virale invasione (s) nel tessuto a livello cellulare 2. Per gli studi di HSV infezioni della pelle o della mucosa, topi, conigli e cavie sono stati utilizzati come modelli animali. Infezione della pelle è stato istituito con iniezione intradermica o graffiare la pelle in presenza del virus, e lo sviluppo della malattia è stata correlata con la produzione di virus. Questi metodi hanno aiutato a comprendere vari aspetti della patogenesi della malattia, e sono utilizzate per valutare farmaci antivirali. Per studiare infezione da HSV al tissue livello, sono stati applicati modelli organotipica di pelle umana. Poiché il tasso di infezione è limitato in queste culture zattera, solo un numero limitato di studi che hanno valutato l&#39;infezione, diffusione virale e gli effetti dei componenti antivirali sono stati pubblicati 3-6. Al fine di caratterizzare i determinanti cellulari che svolgono un ruolo durante HSV-1 nell&#39;epitelio intatto, abbiamo stabilito un protocollo per la ex vivo studi infezioni dell&#39;epidermide murini 7. Pelle è stato preparato da neonati o dalle code di topi adulti. Dal momento che HSV-1 non è riuscito a infettare campioni di pelle completi, che sono stati immersi in mezzo contenente il virus, abbiamo separato l&#39;epidermide dal derma da dispasi trattamento II. Dopo variabile dei fogli epidermica su terreno contenente il virus, cellule infettate possono essere visualizzati nello strato epidermico basale in vari momenti postinfection (pi) 7. Per visualizzare l&#39;inizio di infezione in cellule singole prima replica virale unnd produzione di virus, abbiamo macchiato con un anticorpo contro la proteina delle cellule infettate 0 (ICP0), che è una delle prime proteine ​​espresse durante HSV-1. La localizzazione cellulare di ICP0 passa attraverso fasi distinte durante l&#39;infezione precoce. Mentre ICP0 è presente in foci nucleari in una fase precoce di espressione genica virale, rilocalizzazione di ICP0 al citoplasma indica una successiva fase di infezione 8. Abbiamo usato la ex vivo di infezione test di fogli epidermica da diversi modelli murini per testare il potenziale ruolo di diversi fattori cellulari durante l&#39;infezione. Per affrontare l&#39;impatto di Rac1 come un regolatore chiave delle dinamiche di actina, abbiamo infettati l&#39;epidermide di topi con delezione specifica per cheratinociti del gene Rac1 9. Questo modello ha permesso di studiare le conseguenze delle carenti Rac1 sull&#39;efficienza di HSV-1 negli strati epidermici cheratinociti. Il confronto di epidermide infette del controllo littermates ririvelò alcuna differenza significativa, indicando che l&#39;assenza di Rac1 non ha avuto effetto sulla apertura di infezione nello strato basale dell&#39;epidermide 7. L&#39;utilizzo di ulteriori modelli murini ci ha permesso di affrontare le quali recettori cellulari mediano ingresso nell&#39;epidermide. Infettare fogli epidermica sia da nectin-1 o topi HVEM-deficienti con HSV-1 ha rivelato che l&#39;ingresso del virus iniziale in tessuto dipende fortemente dalla presenza di nectin-1 10. Inoltre, i nostri risultati dimostrano che HVEM può anche servire come recettore nell&#39;epidermide murini, anche se meno efficiente rispetto nectin-1 10. Per affrontare la distribuzione spaziale delle cellule infettate negli strati epidermici, visualizziamo espressione ICP0 in sezioni di tessuto e epidermiche monti interi (Figura 1). In criosezioni di pelle completa, nessuna cellula-ICP0 esprimono vengono rilevati (Figura 1). Al contrario, criosezioni di fogli epidermiche dimostrano citoplasmaticaEspressione ICP0 nello strato basale a 3 ore già pi (Figura 1). In tempi più recenti, la diffusione virale di soprabasali strati può essere visualizzato. La distribuzione spaziale delle cellule infette nello strato basale può essere facilmente seguita in epidermiche supporti interi (Figura 1). Al momento l&#39;infezione da HSV-1 a 100 PFU / cellula, circa il 50% dei cheratinociti basali dell&#39;epidermide interfollicolari mostrano espressione ICP0 a 1,5 ore pi A questo punto momento cellule più infettate esprimono ICP0 nucleare. La rilocalizzazione della ICP0 nel citoplasma che indica una fase successiva dell&#39;espressione genica precoce è presente in quasi tutte le cellule a 3 ore pi (Figura 1). Queste modalità di visualizzazione cellule infette su ex vivo HSV-1 forniscono un potente test per studiare l&#39;effetto degli inibitori o di componenti cellulari mutate cancellati / su ingresso del virus e la diffusione nel tessuto.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/high glucose/GlutaMAX</td>
			<td>Life Technologies</td>
			<td align="right">31966047</td>
			<td>needed for cultivation of epidermal sheets</td>
		</tr>
		<tr>
			<td>dispase II powder</td>
			<td>Roche</td>
			<td align="right">4942078001</td>
			<td>has to be solved in heated PBS</td>
		</tr>
		<tr>
			<td>enzyme-free cell dissociation solution</td>
			<td>Sigma</td>
			<td>C5914</td>
			<td>needed for very gentle dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>TrypLE select cell dissociation solution</td>
			<td>Life Technologies</td>
			<td>12563-029</td>
			<td>needed for dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>chelex 100 resin</td>
			<td>Bio-Rad</td>
			<td>142-2832</td>
			<td>needed for chelation of polyvalent metal ions from the fetal calf serum</td>
		</tr>
		<tr>
			<td>gelatin from cold water fish skin&#160;</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td>needed for minimization of non-specific antibody binding</td>
		</tr>
		<tr>
			<td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-11029</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-21429</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (conc.: 0.1 mg/ml)</td>
			<td>Sigma</td>
			<td align="right">36670</td>
			<td>used to counterstain the nucleus</td>
		</tr>
	</tbody>
</table>

ex vivo infection of murine epidermis with herpes simplex virus type 1

Per inserire il suo ospite umano, il tipo di virus herpes simplex 1 (HSV-1) deve superare la barriera della mucosa superfici, pelle, o cornea. HSV-1 obiettivi cheratinociti durante l&#39;inserimento iniziale e stabilisce una infezione primaria nell&#39;epitelio, che è seguita da infezione latente di neuroni. Dopo la riattivazione, i virus possono diventare evidente a siti mucocutanee che appaiono come vescicole cutanee o ulcere della mucosa. Come HSV-1 invade la pelle o le mucose e raggiunge i suoi recettori è poco conosciuta. Per studiare il percorso invasione HSV-1 nel tessuto epidermico a livello cellulare, abbiamo stabilito un modello ex vivo infezione dell&#39;epidermide murine, che rappresenta il sito di infezione primaria e ricorrente in pelle. Il saggio comprende la preparazione della pelle murina. L&#39;epidermide è separato dal derma mediante trattamento dispasi II. Dopo galleggiante i fogli epidermica su terreno contenente il virus, il tessuto è fisso e l&#39;infezione può essere visualizzato in diversi momenti postinfection bycolorazione delle cellule infettate con un anticorpo contro la HSV-1 immediato proteina ICP0 presto. ICP0-esprimenti cellule può essere osservato nello strato basale cheratinociti già a 1,5 ore postinfection. Con tempi più lunghi di infezione, le cellule infettate vengono rilevati in strati soprabasali, indicando che l&#39;infezione non si limita ai cheratinociti basali, ma il virus si diffonde ad altri livelli nel tessuto. Utilizzando fogli epidermico di vari modelli di mouse, il protocollo di infezione consente di determinare il coinvolgimento delle componenti cellulari che contribuiscono alla HSV-1 invasione nei tessuti. Inoltre, il saggio è adatto a testare inibitori nel tessuto che interferiscono con le fasi iniziali di entrata, diffusione cellula-cellula e la produzione di virus. Qui, descriviamo il protocollo ex vivo infezione in dettaglio e presentiamo i nostri risultati con i topi Nectin-1 o HVEM-carenti.

La sfida del metodo è quello di preparare fogli epidermici in cui HSV-1 possono penetrare dallo strato basale. La fase critica è la separazione dell&#39;epidermide dal derma mediante trattamento dispasi II, che, a seconda del ceppo di topo, deve essere adattato. La concentrazione di dispasi II può variare da 2.5 a 5 mg / ml, e il tempo di incubazione da 20 a 45 min. La colorazione del filamento intermedio proteina cheratina 14 permette facilmente predire se lo strato epidermico basale può essere infettati o se mostr...

Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex Vivo infezione di Murine epidermide con Herpes Simplex Virus di tipo 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

ex-vivo-infection-of-murine-epidermis-with-herpes-simplex-virus-type-1

Research

JoVE Journal

Immunology and Infection

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

11.1K Views.  University of Cologne. The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Video: Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Imaging ex vivo di cellule T nel Fette murini Linfonodo con grandangolari e microscopi confocale

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Ricerca

Immunologia e infezioni

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

A primaria del sistema cultura Neuron per lo Studio della latenza del virus Herpes Simplex e la riattivazione

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Trans-vivo Delayed Type Ipersensibilità Assay per Antigen regolamento specifico

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Valutazione della efficacia e la tossicità degli RNA targeting HIV-1 Produzione per l&#39;uso in gene terapia farmacologica

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivo Infezione della Mucosa genitale umana del tessuto linfoide e donna con il Virus dell'immunodeficienza umana 1 e Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modello di cultura Bursal-cellula primaria di pollo Ex Vivo per studiare la patogenesi Virus bursite infettiva

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Analisi temporale della traslocazione nucleare--citoplasmico di Herpes Simplex Virus 1 proteina mediante microscopia confocale immunofluorescente

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Un modello avanzato di murine per la steatoepatite non alcolica in associazione con il diabete di tipo 2

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Test ELISA in vitro per valutare la potenza del vaccino contro la rabbia

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...