Questo studio è stato supportato da Max McGee Nazionale delle Ricerche Centro Giovanile Diabetesat Medical College del Wisconsin e bambini Research Institute of Wisconsin. I finanziatori avevano alcun ruolo nel disegno dello studio, la raccolta el&#39;analisi dei dati, o la preparazione del manoscritto.

1. Prima di impostare un test di soppressione, si ha la necessità di perline cappotto tosylactivated con anti-CD3 umano (clone UCHT1, 1μg/ml concentrazione finale) per la stimolazione delle cellule e poi verificare se le perle sono rivestiti in modo efficiente attraverso la creazione di un test in vitro proliferazione utilizzando umano cellule T <ol><li> Prendere 1 ml di M-450 perle tosylactivated dal flacone originale, mettere in stand magnetici e tenere premuto fino a quando tutte le perle hanno aderito al lato del tubo. Rimuovere il tubo di buffer mentre è ancora in stand magnetico. Prendete del tubo di supporto magnetico e aggiungere 1 ml di buffer1, posizionare il tubo in stand magnetico di nuovo e togliere il tubo buffer1 mentre è in stand magnetici; perline risospendere in 1 ml di buffer1 e aggiungere 40μl di anti-CD3 umano, agitare a 37 ° C per 15 minuti, aggiungere 0,1% w / v BSA e continuare agitazione per le prossime 16 ore. </li><li> Lavare le perline in buffer2 due volte per 5 minuti a 2-8 ° C e una volta in buffer3 per 5 minuti a 2-8 ° C con supporto magnetico, come spiegato sopra; rimuovere il buffer e risospendere le sfere in 1ml di buffer2, le perle sono 4x10 concentrazione 8 / ml finale e pronto per l&#39;uso. </li><li> Aliquota 50.000 e 25.000 PBMC / e in triplice copia in una piastra da 96 pozzetti e aggiungere numero variabile di perle rivestite CD3 (4x10 8 perline / ml, CD3 1μg/ml, ad esempio 1, 2, 3, 4, 5 perle / cella ) al fine di determinare il numero ottimale di perle per cella. Dopo 72 ore di cultura, aggiungere 1μCi [3 H] timidina e continuare incubazione a 37 ° C per le prossime 16 ore. Celle di raccolta in lamiera raccolta Multisala (Millipore), aggiungere liquido di scintillazione e leggere i conteggi per minuto (cpm) / bene usando Top Conte NXT (Packard, CT). Utilizzare le perline / rapporto cellulare quando CPM sono superiori a 5000 ma inferiore a 15000 per evitare iperstimolazione Tregs, che potrebbe perdere la funzione soppressiva. Di solito, rapporto di 3 perle / delle cellule stimola sia responder e le cellule Treg in tutti i gruppi di soggetti testati finora 1-4. </li></ol> 2. Isolamento PBMC da sangue intero da donatori sani o da leukopacks umano o buffy-coat (BC) di solito tratto da volontari sani e disponibili gratuitamente presso Centri locali Trasfusionale (Figura 1) <ol><li> Diluire il BC (~ 50 ml) 1:6 con PBS (aggiungere 250 ml). Ora c&#39;è 300ml volume totale. Lentamente strato 25ml di BC diluito in cima 15ml Ficoll-Paque PLUS aggiunto a tubi Falcon da 50 ml, senza disturbare gli strati. Centrifugare a 800xg (1400rpm in una centrifuga Sorvall con rotore oscillante SH-3000) per 30 minuti a 4 ° C, con freni spento. </li><li> Raccogliere accuratamente lo strato PBMC (fase intermedia) e trasferirlo in una nuova tubi Falcon da 50 ml. Lavare PBMC compilando i tubi fino a 50 ml con DPBS. Raccogliere due pellet cellulari in un tubo. Centrifugare a 400xg per 10 minuti a 4 ° C. Ripetere il lavaggio passo due volte, ogni volta che combina due pellet cellulari in un unico tubo. Unire tutti gli pellet cellulari in un unico tubo 50ml. </li><li> Conte PBMC mediante test di esclusione tripan blu. Effettuare una diluizione 1:10 in Tricloroetano aggiungendo 20μl di PBMC di 180μl di Tricloroetano macchia. Mescolare bene e prendere 20μl a contare in emocitometro immediatamente. Contare i numeri di tutte le cellule senza macchia e solo blu macchiato situati in due blocchi quadrati ciascuno contenente 16 quadrati più piccoli. Prendere la media di entrambi i numeri e moltiplicare per 10. Dividere questo numero per 100 per ottenere il numero di PBMC / ml. Percentuale di cellule vitali calcolare come [1 - (numero di cellule blu / numero di cellule totali) x100]. Procedere, se la vitalità è ≥ 95%. </li></ol> 3. MACS pre-tipo di cellule T CD4 <ol><li> Prima di procedere, il trasferimento di 1ml di PBMC in un tubo nuovo, aggiungere 4 ml di media per preparare le cellule per irraggiamento con 5000rad-questi saranno cellule presentanti l&#39;antigene (APC). </li><li> Centrifugare il resto delle celle a 250xg nel buffer PBS/2mM EDTA/0.5% BSA per 10 minuti a 4 ° C. Eliminare le cellule surnatante e risospendere in 4 ml di tampone PBS/2mM EDTA/0.5% BSA. </li><li> Aggiungere 200μl di MACS microsfere anti-CD4 e incubare a 4 ° C per 20 minuti. </li><li> Lavare con l&#39;aggiunta di 40 ml di tampone PBS/2mM EDTA/0.5% BSA e centrifugare a 250xg per 10min a 4 ° C. Eliminare il surnatante e risospendere in 8ml di degassata, temperatura ambiente PBS/2mM EDTA/0.5% buffer di BSA. </li><li> Filtro separato la sospensione cellulare con pre-filtri di separazione prima di caricarli su una colonna LS. </li><li> Dividere la sospensione cellulare e 4ml strato di ogni colonna LS oltre calibrato (con 3 ml di tampone deggassed PBS/2mM EDTA/0.5% BSA). Colonna LS è stato risolto nel separatore MidiMACS. Prima di sospensione cellulare si esaurisce, o rieseguire eluato o aggiungere 3 ml di temperatura ambiente deggassed PBS/2mM tampone EDTA/0.5% BSA e lasciare scorrere il buffer. Aggiungere più di buffer fino a quando non viene fuori chiara. </li><li> Pipettare 5 ml di buffer deggassed sulla colonna LS, rimuovere colonna LS dal separatore MidiMACS e posto in una nuova provetta sterile raccolta 15ml lasciar correre ~ 1,0 ml attraverso. Mettere lo stantuffo nella colonna e spingere lentamente il resto delil volume in uscita. </li><li> Fate lo stesso con entrambe le colonne LS e combinare le due frazioni CD4 +. Aggiungi fino a 50ml di PBS e contare le cellule. Rendimento atteso è fino a 5x10 8 cellule. Centrifugare a 400xg per 10 minuti e risospendere le cellule bene in 2 ml di buffer PBS/2mM EDTA/0.5% BSA. </li></ol> 4. Fluorescente attivato cella Ordinamento (FACS), isolamento (Figura 2) <ol><li> Fai un cocktail di anticorpi marcatori CD per i seguenti marcatori superficie cellulare (tenere al riparo dalla luce): 20 ml di anti-umano CD8-FITC (clone RPA-T8), 20 ml di anti-umano CD14-FITC (clone M5E2; LPS recettore), 20 ml di anti-umano CD32-FITC (clone FLI8.26; FcγR di tipo II) e 6 ml di anti-umano CD116-FITC (clone M5D12; GM-CSFRα catena) e, in alternativa, aggiungere l&#39;anti 40μl umani CD4-APCCy7 (clone RPA-T4). </li><li> Prendete 5μl dalla sospensione cellulare 2ml e macchia con 2μl di cocktail macchia, questo è un tubo per la determinazione della soglia (Fluorocromo Minus One-FMO) 5. </li><li> Aggiungere 50 ml di anti-umano CD25-PE (clone M-A251, IL-2Rα) al cocktail macchia, e aggiungere il cocktail di sospensione cellulare e incubare a 4 ° C 30 minuti. Lavare le cellule in tampone PBS, centrifugare a 400xg per 10 minuti e risospendere le cellule ad una concentrazione di 10 7 cellule / ml. </li><li> Preparare le cellule senza macchia e cellule o perline colorate con solo fluorocromo da usare come controllo della compensazione per l&#39;ordinamento delle cellule di FACS Aria (BD Biosciences, San Jose, NJ). </li><li> Acquisire le cellule nel tubo FMO che permetterà all&#39;utente di impostare la soglia per l&#39;ordinamento delle cellule T CD25 + (Figura 2a). Impostare un cancello intorno FITC-negativi cellule per escludere FITC cellule positive composto da monociti, macrofagi e tutti gli altri CD4 +-non-cellule T (Figura 2b). </li><li> In un grafico distinto, disegnare cancelli per CD25-, CD25low e CD25high cellule T-Tregs (top 1% delle cellule che esprimono il maggior numero di CD25) (Figura 2c). Sottoinsiemi di cellule mostrano tipicamente elevata purezza (figura 2d, 2e e 2f). </li><li> Centrifuga provette con celle a 400xg per 10 minuti e tenerli in ghiaccio fino placcatura. </li></ol> 5. Istituito colture cellulari in piastra da 96 pozzetti (schema allegato come Tabella 1) in 200μl/well <ol><li> 50μl aliquota di CD3 rivestite perle (1 mg / ml) calcolato da 3 perline / cella responder in un pozzo, risospesi in mezzi di comunicazione completa con il 10% pool di siero umano AB in U-bottom piastre a 96 pozzetti. (Ad esempio, per rendere i media 2ml con CD3 rivestita, prendere 7.5μl dal magazzino del CD3 rivestite perle-finale 4x10 concentrazione 8 / ml e diluire in 2 ml di media, ogni 50μl conterrà 75.000 beads-3beads/cell). </li><li> Diluire APC irradiati concentrazione cellulare di 5x10 5 / ml e aggiungere 50μl (conterrà 2,5 x 10 4 cellule) in ciascun pozzetto con la stimolazione aggiunto in precedenza, tra cui pozzi etichettato come &quot;Tregs solo&quot;, &quot;APC solo&quot; e &quot;media solo&quot; nella tabella 1. </li><li> Aggiungere 2,5 x 10 4 / pozzetto CD4CD25 o CD4CD25low cellule T in triplicato a seguito di progettazione nella tabella 1. </li><li> Aggiungi Tregs di co-culture (riga B, Tabella 1) nel rapporto 1:10 (2.500 cellule Treg) e di pozzi etichettati come &quot;Tregs solo&quot; e incubare la piastra a 37 ° C in CO 2 incubatore al 5% di CO 2 , satura di umidità per 72 ore. </li><li> Pulse pozzetti con 1μCi [3 H] timidina e continuare l&#39;incubazione a 37 ° C per la prossima 16 ore. </li></ol> 6. La raccolta e il conteggio <ol><li> Celle di raccolta sul piatto raccolta multischermo con Packard filtermate mietitrice o sistema alternativo. </li><li> Aggiungere liquido di scintillazione (Microscint 20), la raccolta di copertura piatto con coperchio in plastica trasparente, in preparazione per la fase finale. </li><li> Leggi conteggi per minuto (cpm) / bene usando Top Conte NXT (Packard, CT) o un sistema alternativo. </li></ol> 7. Percentuale di calcolo di soppressione <ol><li> Le cellule sono state coltivate in triplicato, medio è calcolato per ogni condizione. Se il coefficiente di variazione è&gt; 30%, il valore anomalo è eliminato e solo cpm da due pozzi sono nella media. Percentuale di repressione si ottiene calcolando [(sc) / s] x 100%, dove s = cpm in singola cultura e c = cpm in co-coltura. </li><li> Come ingenuo (CD25-) e in vivo attivato (CD25low) cellule T effettrici sono placcati come responder cellule T, la differenza nella capacità di Tregs di sopprimere ciascuno di questi sottoinsiemi è catturato e usato come indicatore prognostico potenziale funzionale sulla base del presupposto che le cellule attivate sono più difficili da sopprimere. </li></ol> 8. Rappresentante dei risultati: Grande variabilità nei metodi utilizzati e dei risultati derivanti da test in vitro soppressione umano ci hanno spinto a effettuare uno studio completo delle condizioni che influenzano l&#39;analisi 1. Abbiamo sviluppato un test che mette alla prova non solo la funzione Treg, ma anche la loro purezza, considerando il basso rapporto tra Tregs: Teffs (1:10), che abbiamo stabilito in precedenza 6. Inoltre, Tregs differiscono in thEIR capacità di sopprimere con successo ingenuo e in vivo-cellule T attivate, anche in soggetti sani, come mostrato nella Figura 3 e nei nostri studi precedenti 2,4, che diventa più evidente se l&#39;equilibrio immunitario è compromesso, come nei soggetti a rischio di sviluppare diabete di tipo 1 . Il test ha funzionato molto bene nello studio dove abbiamo confrontato funzione soppressiva del naturale (nTregs), inducibile (iTregs) e in vitro nTregs espanso, che ci permette di confrontare tra loro funzione di controllo sani, di recente insorgenza (RO) diabete di tipo 1 e di vecchia data (LS ) dei soggetti diabete di tipo 1. Abbiamo concluso che i soggetti RO diabete di tipo 1 ha una migliore capacità di generare funzionale sia iTregs e nTregs ampliato rispetto ai soggetti di controllo LS T1D e sano 7. Pertanto, questo test può essere utilizzato come un ottimo strumento per il riconoscimento di entrambi uno stato precoce e tardivo di squilibrio del sistema immunitario. Schema 1 Rappresentazione schematica delle fasi coinvolte nel test in vitro soppressione <img src="/files/ftp_upload/3071/3071fig1.jpg" alt="Figura 1"/> Figura 1. Fasi del test in vitro soppressione presentata con foto <img src="/files/ftp_upload/3071/3071fig2.jpg" alt="Figura 2"/> Figura 2. Strategia di gating in cella di isolamento FACS. a) CD25 soglia + è stata regolata secondo Fluorocromo Minus One (FMO), b) le cellule sono state gated come FITC-negativi, c) FITC-negativi cellule sono state ulteriormente gated e raccolto come CD + CD25-, CD + CD25low e CD + CD25high ( Tregs) dimostrato con percentuali, d), FACS ordinati Tregs dopo l&#39;ordinamento, e), FACS ordinati CD + CD25low dopo l&#39;ordinamento, ed f) FACS ordinati CD4 + CD25-cellule T dopo l&#39;ordinamento <img src="/files/ftp_upload/3071/3071fig3.jpg" alt="Figura 3"/> Figura 3. I risultati rappresentativi di soggetti sani a) i risultati rappresentante di conteggi al minuto (cpm) dei soggetti sani presenta come singole culture per tutti i sottoinsiemi di cellule coinvolte (ingenuo-CD25-, in vivo attivato-CD25low, cellule presentanti l&#39;antigene-APC e cellule T regolatorie-Treg), così come co-colture di cellule T responder (CD25-o CD25low) e Tregs. b) Percentuale di soppressione di ogni CD25-e CD25low responder cellule T autologhe da Tregs è presentato per i soggetti sani di controllo (n = 4). Soppressione è stato calcolato come [(sc) / s] x 100%, dove s = cpm in singola cultura e c = cpm in co-coltura. Anche se lieve differenza nella capacità di Tregs per sopprimere le cellule T responder è stato notato, non era significativa (paired t-test p = 0,08). C) Presentato sono cpm di soggetti a rischio per ogni singola cultura, tra CD25 e CD25low come responder cellule T e APC e Tregs, e co-culture in cui è seminato ogni cella risponditore T sottoinsieme con Tregs (CD25-/Tregs e CD25low/Tregs). d) Percentuale di soppressione di ogni CD25-e CD25low responder cellule T autologhe da Tregs è presentato per i soggetti a rischio (n = 4). La differenza nella capacità di Tregs per sopprimere CD25-versus CD25low responder cellule T è stata significativa (paired t-test p = 0,04). Tabella 1. Schematica messa a punto di test in vitro soppressione <table border="1"><tbody><tr><td></td><td> 1-3 </td><td> 4-6 </td><td> 7-9 </td><td> 10-12 </td></tr><tr><td> A </td><td> CD4CD25- </td><td> CD4CD25low </td><td> mezzi di comunicazione solo </td><td> mezzi di comunicazione solo </td></tr><tr><td> B </td><td> CD4CD25-/ Tregs </td><td> CD4CD25low / Tregs </td><td> Tregs solo </td><td> APC solo </td></tr><tr><td> C </td><td></td><td></td><td></td><td></td></tr><tr><td> D </td><td></td><td></td><td></td><td></td></tr><tr><td> E </td><td></td><td></td><td></td><td></td></tr><tr><td> F </td><td></td><td></td><td></td><td></td></tr><tr><td> G </td><td></td><td></td><td></td><td></td></tr><tr><td> H </td><td></td><td></td><td></td><td></td></tr></tbody></table>

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Nessun conflitto di interessi dichiarati.

Essendo l&#39;unica caratteristica unica di Tregs, la funzione di soppressione dovrebbero essere testati in modo affidabile e uniformemente tra soggetti nelle diverse fasi di sviluppo della malattia all&#39;interno della stessa e tra studi differenti. Offriamo i dettagli del test di soppressione sviluppato nel nostro laboratorio come il nostro contributo alla standardizzazione di questo test. Nel nostro studio di ottimizzazione ampio, abbiamo determinato che la stimolazione delle cellule T con anti-CD3 umano rivestite p...

<table border="1"><tbody><tr><th> Nome del reagente o strumento </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Ficoll-Paque PLUS </td><td> Amersham Pharmacia Biotech </td><td> 17-1440-03 </td><td></td></tr><tr><td> DPBS-1X </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> Tricloroetano </td><td> Invitrogen </td><td> 15250-061 </td><td></td></tr><tr><td> anti-CD4 microsfere </td><td> Miltenyi </td><td> 130-045-101 </td><td></td></tr><tr><td> Pre-separazione filtri </td><td> Miltenyi </td><td> 130-041-407 </td><td></td></tr><tr><td> LS colonna </td><td> Miltenyi </td><td> 130-042-401 </td><td></td></tr><tr><td> EDTA </td><td> Invitrogen </td><td> 15575-020 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> B4287 </td><td></td></tr><tr><td> Anti-umano CD4-APCCy7 (clone RPA-T4) </td><td> BD Pharmingen </td><td> 557852 </td><td></td></tr><tr><td> Anti-umano CD25-PE (clone M-A251, IL-2Rα) </td><td> BD Pharmingen </td><td> 555432 </td><td></td></tr><tr><td> Anti-umano CD8-FITC (clone RPA-T8) </td><td> BD Pharmingen </td><td> 555366 </td><td></td></tr><tr><td> Anti-umano CD14-FITC (clone M5E2; recettore LPS) </td><td> BD Pharmingen </td><td> 555397 </td><td></td></tr><tr><td> Anti-umano CD32-FITC (clone FLI8.26; FcγR di tipo II) </td><td> BD Pharmingen </td><td> 555448 </td><td></td></tr><tr><td> Anti-umano CD116-FITC (clone M5D12; GM-CSFRα catena) </td><td> BD Pharmingen </td><td> 554532 </td><td></td></tr><tr><td> Dynalbeads M-450 tosylactivated </td><td> Invitrogen </td><td> 140-13 </td><td></td></tr><tr><td> Anti-CD3 umano </td><td> Ancell </td><td> 144-024 </td><td></td></tr><tr><td> Buffer1 </td><td> Fatto in casa </td><td></td><td> 0,1 M Na 2 B 4 O 7 pH7.6 </td></tr><tr><td> Buffer2 </td><td> Fatto in casa </td><td></td><td> PBS/2mM EDTA / 0,1% di BSA pH7.4 </td></tr><tr><td> Buffer3 </td><td> Fatto in casa </td><td></td><td> 0,2 M Tris/0.1% BSA pH8.5 </td></tr><tr><td> Completo RPMI dei media </td><td> Fatto in casa </td><td></td><td> RPMI 1640 i media 2 mM L-glutammina 5 HEPES mM 100 U / mg / ml Peni / strept 0,5 mM di piruvato di sodio </td></tr><tr><td> [3 H] timidina </td><td> Perkin Elmer </td><td> NET027Z005MC </td><td></td></tr><tr><td> pool di siero umano AB </td><td> Atlanta Biologicals </td><td> S40110 </td><td></td></tr><tr><td> Raccolta piastra multischermo </td><td> Millipore </td><td> MAHFC1H60 </td><td></td></tr><tr><td> Microscint 20 </td><td> Perkin Elmer </td><td> 6013621 </td><td></td></tr></tbody></table>

human in vitro suppression as screening tool for the recognition of an early state of immune imbalance

Cellule T regolatorie (Tregs) sono mediatori critici della tolleranza immunitaria di auto-antigeni. Inoltre, essi sono regolatori cruciali della risposta immunitaria in seguito ad un infezione. Nonostante gli sforzi per identificare marcatori unica superficie sulla Tregs, l&#39;unica caratteristica unica è la loro capacità di sopprimere la proliferazione e la funzione delle cellule T effettrici. Mentre è chiaro che solo saggi in vitro possono essere utilizzati nella valutazione funzione umana Treg, questo diventa problematica in sede di valutazione dei risultati di studi trasversali in cui le cellule sane e cellule isolate da pazienti con malattie autoimmuni (come il diabete di tipo 1, diabete di tipo 1) è necessario da confrontare. C&#39;è una grande variabilità tra i laboratori del numero e del tipo di risponditore cellule T, la natura e la forza della stimolazione, Treg: rapporti responder e il numero e il tipo di cellule presentanti l&#39;antigene (APC) utilizzati in test di soppressione umani in vitro. Questa variabilità rende il confronto tra studi di misurazione funzione Treg difficile. Il campo Treg ha bisogno di un test standardizzato di soppressione che funzionano bene con entrambi i soggetti sani e quelli con malattie autoimmuni. Abbiamo sviluppato un test in vitro di soppressione che mostra poca variabilità intra-saggio nella stimolazione delle cellule T isolate da volontari sani rispetto ai soggetti con sottostante distruzione autoimmune delle cellule β pancreatiche. L&#39;obiettivo principale di questo pezzo è quello di descrivere un test di soppressione umani in vitro che permette il confronto tra gruppi di soggetti diversi. Inoltre, questo saggio ha il potenziale per delineare una piccola perdita in nTreg funzione e anticipare ulteriormente la perdita per il futuro, individuando così i soggetti che potrebbero trarre beneficio dalla terapia immunomodulante preventiva 1. Di seguito, forniamo la descrizione accurata delle fasi di questa procedura. Speriamo di contribuire alla standardizzazione dei test in vitro soppressione utilizzato per misurare la funzione Treg. Inoltre, offriamo questa analisi come strumento per riconoscere uno stato iniziale di squilibrio immunitario e un potenziale biomarcatore funzionale per diabete di tipo 1.

Watch this Scientific Journal Video about Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance at JoVE.com

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs sono potenti soppressori del sistema immunitario. Vi è una mancanza di marcatori di superficie unica per definire loro, quindi, le definizioni di Tregs sono soprattutto funzionali. Qui descrivere un ottimizzato<em&gt; In vitro</em&gt; Test in grado di identificare squilibrio immunitario nei soggetti a rischio di sviluppare diabete di tipo 1.

Umano In Vitro</emSoppressione> come strumento di screening per il riconoscimento di uno Stato precoce di squilibrio immunitario

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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JoVE Journal

Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

14.4K Views.  Medical College of Wisconsin. Tregs sono potenti soppressori del sistema immunitario. Vi è una mancanza di marcatori di superficie unica per definire loro, quindi, le definizioni di Tregs sono soprattutto funzionali. Qui descrivere un ottimizzato In vitro Test in grado di identificare squilibrio immunitario nei soggetti a rischio di sviluppare diabete di tipo 1.

Video: Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Ricerca

Immunologia e infezioni

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

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

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

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

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

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 ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un<em&gt; In Vitro</em&gt; Aderenza Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

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

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

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

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

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

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

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. ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Creazione di una configurazione high-throughput screening per piccole molecole che modulano c-di-GMP Segnalazione di<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

La determinazione di soppressione del sistema immunitario contro il sistema nervoso centrale di protezione per farmacologici Interventi a demielinizzazione autoimmune

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

L&#39;analisi dei linfociti stravaso utilizzo di un<em&gt; In Vitro</em&gt; Modello del Sangue-Cervello umano Barriera

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Umano In Vitro come strumento di screening per il riconoscimento di uno Stato precoce di squilibrio immunitario