Gli autori desiderano ringraziare i partecipanti allo studio e ai loro genitori. Vorremmo anche ringraziare il Dott. Réjean Lapointe (CHUM Notre-Dame, Montreal, Canada) per l&#39;accesso al suo lettore ELISpot, Dr. Lubo Alexandrov per l&#39;analisi statistica, e Denis Blais, Sandra Caron, Silvie Valois e Martine Caty per esperto tecnico assistenza. Sostenuto da finanziamenti le Fonds d&#39;opération pour les projets de recherche clinique et d&#39;evalution des tecnologie (CHU Sainte-Justine) per HS e PO, per la Fondation Centre de cancérologie Charles-Bruneau, e dal Leukemia &amp; Lymphoma Society of Canada. ISF è stato sostenuto da borse di studio la Fondation CHU Sainte-Justine e le Fonds de la recherche du Québec-santé (FRQS). AJG è stato il destinatario di borse di studio dal Dipartimento di Microbiologia, Immunologia e Malattie infettive, Université de Montréal (Gabriel-Marquis borsa di studio), FRQS, e la Canadian Institutes of Health Ricerca (CIHR). NM è stato supportato da la Fondation CHU Sainte-Justine, la Fondazione Cole, e FRQS.

Questo protocollo di ricerca è stato approvato dal Consiglio di CHU Sainte-Justine, Montreal, Quebec, Canada, dove è stato condotto lo studio Istituzionale valutazione etica. Il consenso informato è stato chiesto e ottenuto da tutti i partecipanti allo studio, i loro genitori o tutori legali. Tutte le procedure eseguite nei giorni 1 e 2 devono essere eseguite in condizioni sterili (cioè sotto una cappa a flusso laminare). Procedure di sicurezza standard per il trattamento del sangue umano devono essere rigorosamente rispettate. 1. Rivestimento delle piastre <ol><li> Permeabilize il difluoruro di polivinilidene (PVDF) membrane in fondo 96 pozzetti MultiScreen IP piastre ELISpot bianco aggiungendo ad ogni pozzetto 20 pl di 35% etanolo per 1 min. Membrane dovrebbero diventare leggermente traslucido. </li><li> Lavare immediatamente i pozzetti 3 volte con 200 ml di 1x tampone fosfato (PBS; 137 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4, 2 mM KH 2 PO 4, pH 7.4) utilizzando un multichannel pipetta o un dispositivo simile. Questo passaggio è fondamentale dato che i residui di etanolo possono influenzare la vitalità cellulare e legame di anticorpo di cattura. Piastre ELISpot sono fragili e devono essere maneggiati con cura: l&#39;uso di un lavatore automatico non è raccomandato. </li><li> Cappotto pozzetti con 10 ml di topo purificato cattura IFN-γ anticorpo anti-umano diluito in 1x PBS ad una concentrazione finale di 10 pg / ml. Coprire le piastre con pellicola trasparente e regolare incubare una notte a 4 ° C. </li></ol> 2. Blocco Plates <ol><li> Svuotare i pozzetti, toccando asciugare e lavare i piatti 5 volte con 200 ml di 1x PBS. </li><li> Riempire i pozzetti con 200 ml di terreno RPMI 1640 completo e incubare le piastre per 2 ore a 37 ° C (blocco passo). </li><li> Lavare le piastre 5x con 200 microlitri di PBS 1x e mantenere i pozzi pieno di PBS 1x finché le cellule sono pronte per essere placcato. </li></ol> 3. Cella placcatura e di stimolazione <ol><li> Preparare peripheral cellule mononucleate del sangue (PBMC) a partire da campioni di sangue venoso umano mediante centrifugazione su gradienti di Ficoll-Paque utilizzando le procedure standard. In alternativa, utilizzando metodi standard, disgelare le aliquote di PBMC congelati in azoto liquido. Dopo centrifugazione ultimi, risospendere le cellule ad una concentrazione finale di 2 x 10 6 cellule / ml e incubate overnight a 37 ° C sotto atmosfera di CO 2 5% in un incubatore a camicia d&#39;acqua. </li><li> Rimuovere PBMC dall&#39;incubatore e risospendere in un volume finale di 5 ml di RPMI completo 1640. </li><li> Incubare PBMC in presenza di 10 ml di endonucleasi geneticamente da Serratia marcescens per 5 min a temperatura ambiente. </li><li> Rimuovere un&#39;aliquota 50 microlitri di PBMC e mescolare con 50 ml di trypan colorante blu. Contare le celle e stimare% vitalità usando un emocitometro e l&#39;esame microscopico (metodo esclusione del colorante). Vari metodi automatizzati per il conteggio e la valutazione della vitalità cellulare can al cellularecosì essere utilizzato. PBMC deve essere&gt; 95% praticabile. Scartare campione quando vitalità è inferiore al 95%. </li><li> Centrifugare PBMC a 700 xg per 5 minuti, il surnatante scarto, e risospendere le cellule in AIM-V mezzo supplementato con 2% (v / v) di siero umano inattivato (HIS) ad una concentrazione finale di 2 x 10 6 cellule / ml. Mantenere a 37 ° C fino miscele di stimolazione sono aggiunti alla piastra. </li><li> Aggiungere ai pozzetti, una goccia alla volta, 100 microlitri della miscela stimolazione appropriata. Tre le miscele di stimolazione devono essere utilizzati come descritto di seguito: <ol><li> Medio uso AIM-V integrato con il 2% (v / v) di siero umano inattivato (IHS) come controllo negativo. </li><li> Utilizzare anti-CD3 anticorpo monoclonale (clone OKT3) diluito in mezzo AIM-V supplementato con 2% (v / v) IHS ad una concentrazione finale di 0,5 mg / ml come controllo positivo). </li><li> Utilizzare antigene VZV, sotto forma di una γ-irradiati (10.000 Gy; 30 min tempo di esposizione) vaccino vivo attenuato contro VZV (1.350 unità formanti placca [PFU] / ml) diluizioneTED 1:200 in AIM-V mezzo supplementato con 2% (v / v) IHS) o 1 mg di una miscela equimolare di 67 peptidi (15 residui amminoacidici) sintetizzato sulla base della sequenza del VZV immediatamente precoce (IE63) fosfoproteina . Controllare l&#39;efficacia irradiazione monitorando l&#39;assenza di segni visibili di effetto citopatico dopo 4 giorni in culture di PBMC. </li><li> Preparare tutti i pozzetti in duplicato. </li></ol></li><li> Aggiungere ai pozzetti, una goccia alla volta, 100 microlitri di cellule preparate nel passo 5.4 ai pozzetti appropriati. Due pozzetti devono essere lasciati senza cellule (controllo negativo). Incubare per 20 ore a 37 ° C sotto atmosfera di CO 2 5% in un incubatore a camicia d&#39;acqua. Non scuotere, spostare o impilare i piatti su uno sopra l&#39;altro durante l&#39;incubazione. </li></ol> 4. Spot Sviluppo e rilevamento <ol><li> Scartare le cellule e lavare le piastre 10x con PBS 1x integrato con 0,05% (v / v) di Tween 20 (PBST). Tween 20 aiuta staccare cellule che hanno aderito al PVDF membrane seguente notte di incubazione. </li><li> Usando una pipetta multicanale, aggiungere 100 ml di 0,5 mg / ml biotina coniugato anti-IFN-γ anticorpo monoclonale (clone 4S.B3) diluito in 1x PBS contenente 0,5% (w / v) di siero albumina bovina (BSA). Incubare le piastre per 2 ore a temperatura ambiente. </li><li> Lavare i pozzetti 6x con PBST e aggiungere ogni ben 100 ml di streptavidina conjugatd con fosfatasi alcalina diluito 1:1000 in 1x PBS contenente 0,5% (w / v) BSA. Coprire le piastre e incubare per 1 ora a temperatura ambiente. </li><li> Lavare le piastre 3x con PBST e 3x con 1x PBS. Aggiungere 100 pl di un fosfato 5-bromo-4-cloro-3-indolil (BCIP) / nitro blu tetrazolio cloruro (NBT) soluzione di substrato e incubare 5 min a temperatura ambiente. </li><li> Lavare le piastre sotto acqua distillata. Rimuovere la sezione inferiore della piastra ELISpot e lavare entrambi i lati della membrana con acqua distillata. Far asciugare i piatti. </li><li> Esaminare i pozzi ed enumerare le macchie con un d stereoscopicamicroscopio issection o la scansione delle piastre utilizzando un contatore posto automatizzato. Conservare le piastre lontano dalla luce se non possono essere esaminati lo stesso giorno. <ol><li> Calcolare la media dei numeri di punti contati in corrispondenti pozzetti duplicati e sottrarre il numero di punti contati in pozzetti di controllo negativo (nessuna cella) dal numero di punti contati nei pozzetti. </li><li> Esprimere la produzione di IFN-γ, come il numero di unità in loco di formatura (SFU) per 10 6 PBMC. </li><li> Si consideri che i campioni di prova sono positivi se il numero di SFU è&gt; 50 per 10 6 PBMC e 2 deviazioni standard (SD) sopra il controllo negativo (medium cellule + AIM-V integrato con il 2% (v / v) IHS). </li><li> Utilizzare un valore di 400 SFU per bene quando i punti sono troppi per essere contati. </li></ol></li><li> Verificare se i dati ELISpot sono distribuiti normalmente o non utilizzare il test di Kolmogorov-Smirnov. Quando i dati sono distribuiti normalmente, effettuare confronti statistici con t tes di Studentt (2 gruppi) o ANOVA e il post-test di Bonferroni (confronti multipli). Se i dati non sono distribuiti normalmente, utilizzare il test di Mann-Whitney U (2 gruppi) o il test di Kruskal-Wallis e post-test di Dunn (confronti multipli). </li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Modifiche e risoluzione di problemi: saggi IFN-γ ELISpot sono stati utilizzati per esaminare le risposte immunitarie mediate da cellule contro una varietà di patogeni microbici, inclusi tipo virus dell&#39;immunodeficienza umana 1 (HIV-1) 24,25, virus dell&#39;epatite C (HCV) 26, 27, e Mycobacterium tuberculosis 28,29, solo per citarne alcuni. Qui abbiamo descritto lo sviluppo di un ELISpot test IFN-γ per misurare l&#39;immunità cellulare contro, con la speranza di definire ...

La prima volta nel 1989 UCBT è sempre più utilizzato come parte del trattamento di vari disturbi del sangue neoplastiche e non neoplastiche in bambini 1. VZV è un alphaherpesvirus umana citopatico che provoca due malattie diverse, varicella (dopo l&#39;infezione primaria) e dell&#39;herpes zoster (dopo la riattivazione). Dopo l&#39;infezione primaria, VZV persiste per tutta la vita dell&#39;ospite riparata entro nervi sensoriali del gangli spinali. Una delle complicanze infettive più minacciosi seguenti UCBT è associato con VZV 2-4. Nel nostro centro clinico, in assenza di VZV la profilassi, l&#39;incidenza cumulativa di malattia VZV malattia da VZV a 3 anni postUCBT era del 46% 2. In questi pazienti, de novo infezione con o riattivazione di VZV è spesso associato con diffusione viscerale al sistema nervoso centrale, polmoni e fegato 5-7. Come profilassi conseguenza, aciclovir, valaciclovir e famciclovir viene comunemente somministrato a UBCT destinatari 8,9. Tuttavia, questa strategia di trattamento non tiene conto del potenziale protettivo di VZV linfociti T specifici o la cinetica di ricostituzione del VZV specifiche risposte delle cellule T. Potenziali problemi associati con l&#39;uso crescente di profilassi antierpetico lungo termine includono a) overtreatment paziente; b) lo sviluppo di resistenza ai farmaci antivirali 10,11; c) violazione di VZV specifico ricostituzione immunitaria 12,13. Poiché il rilevamento di VZV funzionali specifici linfociti T è correlata con la presenza di una protezione a lungo termine da VZV e un miglior risultato clinico 4,14,15, monitoraggio delle cellule mediata risposte immunitarie contro VZV nel periodo post-trapianto potrebbe comportare un uso più razionale antivirale trattamento consentendo medici di distinguere i pazienti che potrebbero trarre vantaggio da VZV profilassi da coloro il cui sistema immunitario è in grado di controllare la replicazione VZV 4,13. Il ELISpot saggio IFN-γ è ampiamente usato per le cellule monitoraggio risposte immunitarie mediate in una varietà di sistemi sperimentali e condizioni cliniche. Macchie sono generate a seguito clivaggio di un substrato cromogenico, generando un precipitato visibile e stabile al sito di reazione. Ogni singolo spot rappresenta quindi l&#39;impronta di una cella producenti citochine individuale. IFN-γ ELISpot non solo misura la capacità delle singole cellule ex vivo per produrre IFN-γ in risposta alla stimolazione in vitro con antigene affine, ma fornisce anche una stima della frequenza di rispondere cellule in una data popolazione cellulare 16,17. Oltre alla sua elevata sensibilità, IFN-γ ELISpot è semplice da eseguire, rendendo possibile il suo impiego nel contesto di protocolli clinici personalizzati volti a guidare l&#39;inizio o cessazione del trattamento antivirale. La procedura di seguito dettagliata descries un saggio ELISpot che è specificamente progettato per rilevare e misurare la produzione di IFN-γ da parte delle cellule mononucleate del sangue periferico dopo stimolazione in vitro con antigeni derivati ​​VZV.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leucocep tube</td>
			<td style="width:101px;">VWR</td>
			<td style="width:143px;">89048-936/89048-932</td>
			<td style="width:397px;">12 ml or 50 ml tubes may be used depending on the volume of blood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ficoll-Paque</td>
			<td style="width:101px;">GE Healthcare</td>
			<td style="width:143px;">17-1440-02</td>
			<td>Protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzonase nuclease</td>
			<td style="width:101px;">Novagen</td>
			<td style="width:143px;">70746-3</td>
			<td>Keep at -20 C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MultiScreenHTS-IP Filter Plate</td>
			<td style="width:101px;">Millipore</td>
			<td style="width:143px;">MSIPS4W10</td>
			<td>Sterile with pore size of 0.45 &#181;m.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse anti-human IFN-&#947; capture antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">551221</td>
			<td>NIB42 clone.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pepmix VZV IE63&#160;</td>
			<td style="width:101px;">JPT Peptide Technologies</td>
			<td style="width:143px;">PM-VZV-IE63</td>
			<td>Dissolve contents of one vial in 40 &#956;L of DMSO. Use within 6 months.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotin-conjugated anti-IFN-&#947; monoclonal antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">554550</td>
			<td>4SB3 clone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptavidin conjugated with alkaline phosphatase&#160;</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-3554</td>
			<td>Dilute for use on the same day.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BCIP/NBT</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-6432</td>
			<td>Protect from light.</td>
		</tr>
	</tbody>
</table>

development of an ifn-&#947; elispot assay to assess varicella-zoster virus-specific cell-mediated immunity following umbilical cord blood transplantation

Varicella zoster (VZV) è una causa importante di morbilità e mortalità dopo trapianto di sangue del cordone ombelicale (UCBT). Per questo motivo, la profilassi antierpetico è amministrato sistematicamente ai destinatari UCBT pediatrici per evitare complicazioni associate con infezione VZV, ma non vi è un consenso prove basate forte che definisce la sua durata ottimale. Poiché cellule T mediata è responsabile del controllo di infezione VZV, valutare la ricostituzione di VZV specifiche risposte delle cellule T seguente UCBT potrebbe fornire indicazioni sul fatto profilassi possono essere mantenute o possono essere sospesi. A tal fine, un VZV specifico interferone gamma (IFN-γ) immunospot enzimatico (ELISpot) saggio è stata sviluppata per caratterizzare la produzione di IFN-γ da linfociti T in risposta alla stimolazione in vitro con vaccino vivo attenuato irradiato VZV. Questa analisi fornisce una misurazione rapida, riproducibile e sensibile di VZV specifico cell immunità adatto per monitorare la ricostituzione del VZV dell&#39;immunità specifica in ambito clinico e di valutare la risposta immunitaria agli antigeni VZV mediata.

Il protocollo ELISpot IFN-γ sopra descritto è stato sviluppato ed ottimizzato nel nostro laboratorio per misurare l&#39;entità e la qualità delle cellule risposte immunitarie mediate dirette contro VZV 4. Diverse fonti di antigene VZV possono essere utilizzati per la fase di stimolazione. Questi includono: a) disponibile in commercio dei detergenti estratti inattivati ​​da VZV infettate cellule Vero 18; b) le piscine di sovrapposizione peptidi sintetici da specifiche proteine ​​codifica...

Watch this Scientific Journal Video about Development of an IFN-Î³ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation at Jo

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Generazioni di nuovi saggi funzionali quali interferone gamma (IFN-γ) ELISpot, che rilevano la produzione di citochine a livello di singola cellula e forniscono sia la caratterizzazione quantitativa e qualitativa delle risposte delle cellule T possono essere utilizzati per valutare la risposta immunitaria cellulo-mediata contro varicella zoster virus (VZV).

Sviluppo di un Varicella-Zoster immunità cellulo-mediata specifica-Virus-IFN γ ELISpot test per valutare seguito cordone ombelicale trapianto di sangue

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

development-an-ifn-elispot-assay-to-assess-varicella-zoster-virus

Research

JoVE Journal

Immunology and Infection

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

15.5K Views.  Université de Montréal.  Generazioni di nuovi saggi funzionali quali interferone gamma (IFN-γ) ELISpot, che rilevano la produzione di citochine a livello di singola cellula e forniscono sia la caratterizzazione quantitativa e qualitativa delle risposte delle cellule T possono essere utilizzati per valutare la risposta immunitaria cellulo-mediata contro varicella zoster virus (VZV). 

Video: Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Enzyme-linked Immunospot Assay (ELISPOT): Quantificazione di Th-1 risposte cellulari immunitarie contro antigeni microbici

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Ricerca

Immunologia e infezioni

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Espansione linfociti T citotossici da sangue del cordone ombelicale che Target Citomegalovirus, virus di Epstein-Barr virus, e Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Applicazione clinica della<em&gt; Bella Addormentata</em&gt; Artificiale e cellule presentanti l&#39;antigene per modificare geneticamente le cellule T da sangue del cordone ombelicale e periferico

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolamento di precursori delle cellule B di sottoinsiemi di sangue del cordone ombelicale

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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 ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Un flusso di Adesione saggio per studiare Leucocyte reclutamento a epatici umani sinusoidali Endotelio in condizioni di stress Shear

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

L&#39;isolamento, la differenziazione e quantificazione di Human anticorpo-secernenti cellule B da sanguigno: ELISpot come una lettura funzionale del umorale immunità

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

L&#39;uso di un monociti monostrato test per valutare fagocitosi Fcy recettore-mediata

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Basato sulla fluorescenza neuraminidasi Inibizione Assay per valutare la suscettibilità dei virus influenzali di neuraminidasi Inhibitor classe di antivirali

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Virus di Zika specifico diagnostico dell'epitopo Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...