This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

Tutti gli esperimenti sugli animali sono stati approvati dalla cura e l&#39;uso Comitato animali presso l&#39;Università del Texas Medical Branch. 1. Isolamento delle DC da topi non infetti e WNV infettate <ol><li> Per età e per sesso partita 6-10 settimane di età, wild-type C57BL / 6 (B6) e MyD88 - / - mice. Inoculare per via intraperitoneale (ip) con 500 unità formanti placca (PFU) di WNV NS4B- P38G mutante. Al giorno 3 dopo l&#39;infezione, eutanasia B6 e MyD88 - / - mice con CO 2. Utilizzare i topi non infetti di entrambi i gruppi come controlli. Utilizzare tre topi da ciascun gruppo. </li><li> Pelliccia bagnato sul lato sinistro del mouse sacrificato con etanolo al 70% e tagliato via il pelo lungo il lato sinistro del mouse, circa a metà strada tra le gambe anteriori e posteriori. Tagliare aprire la cavità del corpo. Rimuovere la milza utilizzando le pinze. Posizionare la milza in una capsula di Petri con RPMI. </li><li> Isolare DC milza utilizzando sfere magnetiche anti-CD11c secondole istruzioni del produttore. </li></ol> 2. Purificazione e etichettatura delle cellule T di OTII topi transgenici <ol><li> Harvest una milza da un ingenuo del mouse OTII come descritto al punto 1.2 di cui sopra e omogeneizzare la milza tra le estremità smerigliato di due slitte. Trasferire la sospensione cellulare in una provetta conica da 15 ml, aggiungere mezzo RPMI fino a 14 ml. Sia la sospensione riposare per 5 min e 13 ml di trasferimento ad una nuova provetta conica da 15 ml. </li><li> Isolare le cellule CD4 + T spleniche utilizzando un kit di isolamento di cellule CD4 + T secondo le istruzioni del produttore. </li><li> Trasferire le cellule in una provetta da 50 ml, lavare due volte con PBS. Risospendere le cellule in PBS con albumina sierica bovina 0,5% (BSA) a 1 x 10 6 cellule per ml, e aggiungere 0,5 mmol / ml di CFSE. Incubare a 37 ° C per 10 minuti, al riparo dalla luce. </li><li> Aggiungere 5 ml terreno completo a freddo (RPMI-1640 con siero fetale bovino 10% di calore-inattivato, 1/100 volantibiotici / antimicotici, 1/100 vol L-glutammina e 1 / 1.000 vol di 1.000 x 2-mercaptoetanolo). </li><li> Incubare in ghiaccio per 5 min. Lavare le cellule una volta con PBS, fix 0.2 x 10 6 etichettati cellule CD4 + T in 2% PFA e acquisire su un citofluorimetro. Utilizzare questo esempio per determinare il livello basale di CFSE. Re-sospende il resto delle cellule etichettate in terreno completo. </li></ol> 3. Co-cultura OTII T cellule con DC di topi infettati da WNV <ol><li> Cultura 2 x 10 5 purificati cellule CD4 + T di topi OTII solo o con DC purificate di wild-type o MyD88 - / - mice (2 x 10 4) in un piatto ben 24, con o senza residui OVA 323-339 (1 g / ml) in 1 ml di mezzo completo per pozzetto. </li><li> Incubare le cellule a 37 ° C per 5 giorni. Celle di raccolta, lavare due volte in tampone FACS (PBS con 1% FBS) e fissare in 0,25 ml 2% PFA. Vortex immediatamente. </li></ol> 4. Citometria a flusso Analisi di In Vitro &lt;/ Em&gt; T cellulare adescamento <ol><li> Per l&#39;acquisizione, fare doppio clic sull&#39;icona del software di citometria a flusso. Per una trama densità lineare FSC-A vs linear SSC-A, selezionare i canali da 0 a 200.000 in FSC-A e 0 a 200.000 su SSC-A. Quindi creare un cancello su cellule vive (P2; vedi Figura 1A). </li><li> Per analizzare l&#39;intensità di fluorescenza di CFSE, aprire l&#39;istogramma del canale FL1 e acquisire 20.000 eventi sulla popolazione gated. Impostare un marcatore su campioni di cellule in coltura OTII sola (Figura 1A). </li><li> Acquisire i campioni per le cellule OTII co-coltura con wild-type DC (Figura 1B) o MyD88 - / - DC (Figura 1C) in modo simile. </li></ol> 5. Isolamento e stimolazione di splenociti per citochine saggi <ol><li> Infettare B6 e MyD88 - / - mice con WNV NS4B-P38G mutante utilizzando la stessa procedura descritta in step 1.1. Al giorno 30 dopo l&#39;infezione, ri-sfida superstite topi con 2.000 PFU del wild-type WNV ceppo ip A giorni 8 e 21 di infezione WNV post-primaria o al giorno 4 dopo l&#39;infezione secondaria, eutanasia topi con CO 2 per la raccolta di milza . NOTA: Abbiamo usato 2-3 topi per gruppo, per ogni punto di tempo. </li><li> Fare una sospensione singola cella di splenociti come sopra descritto al punto 1.2 e 2.2 passo. Contare le cellule utilizzando un emocitometro e li ri-sospendere in 10 ml di media completo. </li><li> Diluire le cellule con mezzo completo a 2,5 x 10 6 cellule / ml. Aggiungere 1 ml di splenociti in un tubo micro-centrifuga 1.5 ml. </li><li> Per simulare cellule CD8 + T, diluire NS4B ed E peptidi specifici WNV (SSVWNATTA e IALTFLAV) a 1 mg / ml in DMSO. Per la stimolazione di cellule CD4 + T, diluire NS3 ed E peptidi specifici WNV (RRWCFDGPRTNTILE e PVGRLVTVNPFVSVA) a 1 mg / ml in DMSO. Aggiungere 10 ml di peptidi alle cellule followed da 1 ml di Brefeldina A soluzione. Utilizzare le cellule senza trattamento peptidi come controlli. Mescolare le cellule. </li><li> Punzone due fori con un ago da 18 G nel tappo della provetta. Incubare le cellule a 37 ° C per 5 ore. </li></ol> 6. intracellulare di citochine colorazione <ol><li> Trasferire le cellule in un nuovo tubo micro-centrifuga. Spin celle per 5 minuti a 300-400 xg e versare il surnatante. Aggiungere 1 ml FACS tampone, rotazione 5 min a 300-400 xg e risospendere cellule pipettando con 120 microlitri FACS buffer. </li><li> Aggiungere 2 ml Fc bloccante e incubare cellule a temperatura ambiente per 10 min. Aggiungere 1 ml FACS buffer per ogni provetta. Spin 5 min a 300-400 x g. </li><li> Risospendere le cellule in 300 ml FACS tampone e dividerli in tre provette (circa 0,8 x 10 6 cellule per provetta). Come mostrato nella Tabella 1, la riserva di primo tubo di ciascuna condizione di coltura per ratto IgG-PE colorazione. Per il secondo tubo, aggiungere 3 ml di anti-CD4 APC per CD4 peptidi-trattati cellule, 3 μl di anti-CD8 FITC alle cellule CD8 peptidi-trattati o entrambi gli anticorpi alle cellule senza trattamento peptidi. Utilizzare il terzo tubo di risarcimento colorazione. Per ogni condizione cultura, utilizzare tre provette di cellule colorate con APC-coniugato CD4, CD8 FITC coniugato o in PE etichettati anticorpi CD3 soli (3 ml per provetta) come controlli di compensazione per FL1, FL2 e canali FL4. </li><li> Cellule Vortex brevemente. Lasciare in ghiaccio per 20 minuti e proteggere dalla luce. Aggiungere 1.4 ml di tampone FACS. Spin 5 min a 300-400 xg e versare il surnatante. </li><li> Agitare per disturbare pellet (o brevemente vortex). Risospendere pellet cellulare in 250 ml di fissaggio / permeabilizzazione solutionby pipettaggio. Incubare a temperatura ambiente per 20 min e proteggere dalla luce. </li><li> Aggiungere 1,2 ml di tampone FACS. Spin 5 min a 300-400 XG e risospendere le cellule in 300 microlitri di buffer FACS. NOTA: A questo punto, le cellule possono essere trasferiti ad un laboratorio BL2 per ulteriore trasformazione o conservata in frigorifero e protetto dalla luce per up per tre giorni. </li><li> Aggiungere 500 microlitri FACS buffer. Spin 5 min a 300-400 XG e risospendere le cellule in 500 l di 1x tampone di permeabilizzazione / lavaggio. Spin 5 min a 300-400 x g. </li><li> Risospendere le cellule in 100 microlitri 1x Perm / Wash, aggiungere 3,5 microlitri anti-IFNγ-PE al tubo precedentemente aggiunto con anticorpi per CD4 e / marcatori cellulari o CD8 T in fase 6.3 e 3.5 ml topo IgG-PE nel tubo riservata per il controllo IgG per 25 min in ghiaccio e proteggere dalla luce. Lavare le cellule aggiungendo 1,4 ml di 1x tampone di permeabilizzazione / lavaggio. Spin 5 min a 300-400 x g. Ripetere la fase di lavaggio con l&#39;aggiunta di 1,4 ml di 1x tampone di permeabilizzazione / lavaggio. </li><li> Spin 5 min a 300-400 xg e decantare il surnatante. Lavare le cellule aggiungendo 1,4 ml di tampone FACS e risospendere in 400 microlitri di tampone FACS per acquisizione finale. </li></ol> 7. Citometria a flusso Analisi delle citochine intracellulari colorazione <ol><li> Utilizzare le stesse impostazioni di acquisizione come al punto 4.1. Create un cancello su cellule vive (P2; Figura 2A). Regolare le tensioni con i tubi di compensazione per ogni gruppo. </li><li> Aperti due nuovi dot plot, una per visualizzare i dati raccolti per FL1 vs. FL2 (CD8 vs. IFN-γ), e l&#39;altra è per visualizzare i dati raccolti per FL4 vs. FL2 (CD4 vs. IFN-γ). Acquisire 50.000 eventi per la popolazione gated di ogni campione. </li></ol>

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WNV è un patogeno BL3. A causa delle norme di sicurezza, test immunologici con i campioni WNV-infetti sono spesso limitati dalla disponibilità di attrezzature in BL3 strutture o più lungo e noioso per eseguire. In un recente studio, abbiamo utilizzato due metodi basati citometria a flusso per studiare cellule risposte immunitarie mediate durante l&#39;infezione WNV 5. In entrambi i test, le cellule infettate da WNV sono stati trattati con 1-2% direttamente o con una soluzione di fissaggio / permeabilizzazi...

Virus West Nile (WNV), una neurotropo, plus-rilevati flavivirus, è una minaccia per la salute pubblica emergente. Attualmente, vaccini sono stati approvati per uso umano 1. Un ceppo WNV attenuato, che ha una sostituzione P38G nel non strutturale (NS) di proteine ​​4B, è noto per indurre non letalità nei topi ma citochine superiore innate e le risposte delle cellule T nei topi wild-type di WNV NY99 ceppo 2. I topi immunizzati con il mutante NS4B-P38G stati tutti protetti da una sfida secondario con letale wild-type WNV. Questo suggerisce che il mutante NS4B-P38G ha caratteristiche adatte per un vaccino candidato ideale. I meccanismi con cui il mutante NS4B-P38G induce protezione ad alta immunità adattativa non sono chiaramente ancora capito. Toll-like receptors (TLR), che riconoscono pattern molecolari associati ai patogeni, svolgono un ruolo fondamentale nell&#39;avvio di immunità innata alle infezioni virali. Il percorso di segnalazione nucleo TLR utilizza mieloide risposta differenziazione primaria geNE 88 (MyD88) come scheda primaria 3,4. In un recente studio, la segnalazione MyD88 ha dimostrato di giocare un ruolo importante nello sviluppo delle cellule mediata durante WNV NS4B- P38G infezione nei topi mutanti 5. Le cellule dendritiche (DC) sono una delle più importanti cellule presentanti l&#39;antigene espositrici la capacità unica di avviare le risposte delle cellule T primarie durante l&#39;infezione virale 6,7. Cellule T CD4 + e CD8 + entrambi contribuiscono all&#39;immunità protettiva di lunga durata e sono importanti per la sopravvivenza di accoglienza a seguito di tipo selvaggio WNV infezione 8,9. Due saggi immunologici sono stati usati in questo studio per valutare le funzioni di queste cellule nei topi mutanti infettati NS4B-P38G. In primo luogo, un dosaggio priming di cellule T in vitro è stato utilizzato per confrontare la capacità presentanti l&#39;antigene di DC di wild-type WNV-infetti e MyD88 - / - mice. Per aumentare la sensibilità del test, ingenuo CD4 &lt;sup&gt; + cellule T sono stati isolati da OTII topi transgenici che esprimono uno specifico Vα2 / Vβ5 TCR per l&#39;ovoalbumina pollo (OVA) peptide 323 - 339. DC di WNV topi infetti sono stati purificati, e co-coltura con carboxyfluorescein succinimidil Pasqua (CFSE ) -labeled cellule CD4 + T in presenza di OVA peptide. Dopo 5 giorni di co-coltura, le cellule sono state raccolte e fissate con paraformaldeide (PFA) e analizzati mediante citometria a flusso. Saggi proliferativa, sono state tradizionalmente effettuata mediante incorporazione di 5-bromo-2&#39;-deossiuridina (BrdU) o triziata deoxyriboside timina (3 HTdr) 10. Tuttavia, questi test sono o radioattivi e / o che necessitano di apparecchiature speciali a livello di biosicurezza (BL) 3 strutture, dove vengono condotti studi WNV. L&#39;analisi citofluorimetrica della proliferazione dei linfociti dimezzando seriale dell&#39;intensità di fluorescenza del colorante vitale CFSE è diventato più comunemente usato in saggi immunologici, come il colorante è più stabilee uniformemente incorporati in cellule, rilevata facilmente mediante citometria di flusso, ed è non radioattivo 11. Il test ha anche la capacità di valutare il numero di divisioni cellulari. Uno dei principali vantaggi di questo test in studi WNV è che la fissazione delle cellule infettate con 1-2% PFA possa inattivare WNV 12, che consentirà l&#39;acquisizione dei campioni con un citometro a flusso in un laboratorio BL2. Successivamente, una procedura modificata intracellulare di citochine colorazione (ICS) è stato utilizzato per studiare il ruolo di MyD88 segnalazione nella regolazione del WNV specifiche risposte delle cellule T in NS4B-P38G topi mutanti-infettati. In questo saggio, splenociti isolati da topi infetti sono stati trattati in vitro con WNV peptidi specifici. Brefeldina A inserito mantenere le citochine all&#39;interno della cellula. Dopo 5 ore di incubazione, le cellule sono state raccolte, lavate e colorate per sottogruppi di cellule T. Le cellule sono state poi fissati in PFA, permeabilizzate e colorate per interferone (IFN) -γ e analizzate mediante citometria a flusso.Come con altri flusso-citometria saggio basato, una volta che le cellule vengono trattate con fissazione e tampone di permeabilizzazione contenente PFA, campioni infetti possono essere trasferiti ad un laboratorio BL2 per l&#39;ulteriore elaborazione e analisi. In diversi studi pubblicati, abbiamo usato ICS per misurare le funzioni effettrici delle cellule T in WNV-infetti topi 13,14. Anche se è ben consolidata, uno svantaggio principale di questo saggio è che la procedura è molto lunga e potrebbe essere più tempo quando eseguita all&#39;interno di strutture BL3. Qui, un metodo basato ICS-tube micro-centrifuga ha dimostrato di essere più fattibile, facile procedere e richiede meno tempo quando eseguita all&#39;interno di un laboratorio BL3.

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">RPMI 1640</td>
			<td style="width:153px;">Invitrogen</td>
			<td align="right" style="width:119px;">11875</td>
			<td>warm up at 37C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD11c magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-001</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD4 magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-248</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFSE</td>
			<td style="width:153px;">Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OVA residue 323-339</td>
			<td>Genscript Corporation</td>
			<td>RP10610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Peptides</td>
			<td style="width:153px;">Proimmune</td>
			<td>PC0AD-D</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Brefeldin A solution</td>
			<td style="width:153px;">BD Bioscience</td>
			<td align="right">555029</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Mouse Fc Blocker</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>14-0161-85</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">APC-conjugated CD4&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>17-0041-81</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">FITC-conjugated CD8&#160;</td>
			<td style="width:153px;">e- bioscience</td>
			<td>11-0081-82</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Fixation/Permeabilization Solution</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right" style="width:119px;">554722</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Permeabilization/wash buffer</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right">554723</td>
			<td></td>
		</tr>
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			<td height="58" style="height:58px;">anti-IFNg-PE&#160;</td>
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			<td height="58" style="height:58px;width:216px;">Accuri flow cytometer</td>
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in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

Un virus attenuato West Nile (WNV), una non strutturale (NS) 4B-P38G mutante, indotta citochine superiore innata e le risposte delle cellule T rispetto al wild-type WNV nei topi. Recentemente, mieloide fattore di differenziazione 88 (MyD88) Segnalazione ha dimostrato di essere importante per priming di cellule T iniziale e lo sviluppo delle cellule di memoria T durante l&#39;infezione WNV mutante NS4B-P38G. In questo studio, due flusso metodi basati citometria - una in vitro delle cellule T saggio di adescamento e una citochina colorazione intracellulare (ICS) - sono stati utilizzati per valutare le cellule dendritiche (DC) e funzioni delle cellule T. Nella cella T adescamento test, la proliferazione cellulare è stato analizzato mediante citometria a flusso dopo co-coltura di cellule dendritiche da entrambi i gruppi di topi con estere carboxyfluorescein succinimidil (CFSE) - etichettato cellule CD4 + T di OTII topi transgenici. Questo approccio ha fornito una determinazione accurata della percentuale di cellule proliferanti CD4 + T con migliorata significativamente sensibilità globale rispetto alla tradizioneal saggi con reagenti radioattivi. Un sistema provetta è stato utilizzato in entrambe le procedure di coltura cellulare e citochine colorazione del protocollo ICS. Rispetto al tradizionale sistema basato piastra di coltura tissutale, questa procedura modificata era più facile per eseguire a livello di biosicurezza (BL) 3 strutture. Inoltre, le cellule infette WNV- sono state trattate con paraformaldeide in entrambi i test, che ha consentito un&#39;ulteriore analisi fuori strutture BL3. Nel complesso, questi in vitro test immunologici possono essere utilizzati per valutare in modo efficace risposta immunitaria cellulo-mediata durante l&#39;infezione da WNV.

Nel saggio T cellule adescamento, le cellule CD4 + T CFSE etichettati sono state coltivate con DC purificate dalla wild-type-mutante infetto NS4B-P38G e MyD88 - / - mice in presenza o assenza di peptidi OVA. Cellule T etichettati coltivate solo con o senza OVA per 5 giorni sono stati usati come controlli negativi. Come mostrato nella Figura 1A, le cellule T sono state gated totale per l&#39;analisi di fluorescenza sul canale FL1. Il marcatore è stato istituito sulla base delle app...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

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 ...

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Research

JoVE Journal

Immunology and Infection

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

8.1K Views.  The University of Texas Medical Branch. Two flow cytometry-based methods – 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.

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

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

Multicolor Citometria a Flusso di analisi Cellular risposta immunitaria in macachi Rhesus

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Ricerca

Immunologia e infezioni

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sette passi per Stellate Cellule

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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. ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP come un nuovo approccio per prevenire l&#39;infezione: Preparazione e<em&gt; In vitro</em&gt; Proprietà antimicrobiche di PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modello per la misurazione Risposte immunitarie Malaria nel contesto della coinfezione da HIV

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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

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

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

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

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

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

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 ...