Name: expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus
Uploaded: 2012-05-07T13:00:00
Description: Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

Questo lavoro è stato sostenuto da un L. Dan Duncan collaborativa assegno di ricerca (CMB e EJS), il National Heart, Lung, and Blood Institute (US4HL081007), un Leukemia and Lymphoma Society Clinical Research Scholar premio (CMB), e il National Cancer Institute (RO1 CA06150816; EJS).

1. Cella di isolamento mononucleare (giorno 0) <ol><li> Inserire spike di luer femmina nella porta di uscita della frazione 20% della unità di sangue cordonale, collegare la siringa e rimuovere il sangue. Trasferimento sangue scongelati a 20 ml di RPMI riscaldato in tubo da 50 ml per centrifuga. Risciacquare sangue borsa dei cavi con 5 ml di RPMI e trasferire al tubo da centrifuga stessa. </li><li> Centrifugare le cellule per 10 minuti a 400 x g. Aspirare il surnatante. </li><li> Risospendere le cellule in 20 ml di R...

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	<li>Kennedy-Nasser, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparable+outcome+of+alternative+donor+and+matched+sibling+donor+hematopoietic+stem+cell+transplant+for+children+with+acute+lymphoblastic+leukemia+in+first+or+second+remission+using+alemtuzumab+in+a+myeloablative+conditioning+regimen.">Comparable outcome of alternative donor and matched sibling donor hematopoietic stem cell transplant for children with acute lymphoblastic leukemia in first or second remission using alemtuzumab in a myeloablative conditioning regimen.</a> Biol. Blood Marrow Transplant. 14, 1245-1245 (2008).</li><li>Hanley, P. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cellular+therapy+for+early+cytomegalovirus+infection+after+allogeneic+stem-cell+transplantation+with+virus-specific+T-cell+lines.">Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines.</a> Lancet. 362, 1375-1375 (2003).</li><li>Sili, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+expansion+of+dendritic+cell-primed+polyclonal+human+cytotoxic+T-lymphocyte+lines+using+lymphoblastoid+cell+lines+for+adoptive+immunotherapy.">Large-scale expansion of dendritic cell-primed polyclonal human cytotoxic T-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy.</a> J. Immunother. 26, 241 (2003).</li><li>Bollard, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+manufacturing+practice-grade+cytotoxic+T+lymphocytes+specific+for+latent+membrane+proteins+(LMP)-1+and+LMP2+for+patients+with+Epstein-Barr+virus-associated+lymphoma.">Good manufacturing practice-grade cytotoxic T lymphocytes specific for latent membrane proteins (LMP)-1 and LMP2 for patients with Epstein-Barr virus-associated lymphoma.</a> Cytotherapy. 13, 518 (2011).</li><li>Hanley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+active+virus-specific+T+cells+that+target+CMV,+adenovirus,+and+EBV+can+be+expanded+from+naive+T-cell+populations+in+cord+blood+and+will+target+a+range+of+viral+epitopes.">Functionally active virus-specific T cells that target CMV, adenovirus, and EBV can be expanded from naive T-cell populations in cord blood and will target a range of viral epitopes.</a> Blood. 114, 1958 (1958).</li><li>Hanley, P. 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Blood Marrow Transplant. 14, 1156 (2008).</li><li>Shpall, E. J., Bollard, C. M., Brunstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cord+blood+transplant+therapies.">Novel cord blood transplant therapies.</a> Biol. Blood Marrow Transplant. 17, S39-S45 (2011).</li><li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+(G-Rex.">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex.</a> J. Immunother. 33, 305 (2010).</li></ol>

<html> Attuali strategie volte a controllare le infezioni virali dopo CBT può essere efficace, ma sono associati a tossicità significative, sono costosi, e non conferiscono protezione a lungo termine contro l&#39;infezione in seguito. In effetti, l&#39;uso di alcuni farmaci antivirali possono limitare l&#39;espansione del virus-specifiche cellule T che altrimenti sarebbero protettivo. 14 Un&#39;altra opzione è l&#39;infusione di donatore-derivati ​​virus-cellule T specifiche. Noi e altri hanno dimostrat...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870-076 </td></tr><tr><td> DC supporti </td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA (Medium Click di) </td><td> Irvine Scientific </td><td> 9195 </td></tr><tr><td> Siero umano </td><td> Gemini Bio Prodotti </td><td> 100-110 </td></tr><tr><td> Gas Permeabile Cultureware 18 </td><td> Wilson-Wolf </td><td> 80040S </td></tr><tr><td> IL-2 </td><td> Chirone (TCH Farmacia) </td><td></td></tr><tr><td> IL-12 </td><td> NCI / CTEP </td><td></td></tr><tr><td> IL-15 </td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7 </td><td> R &amp; S </td><td> AFL207 </td></tr><tr><td> IL-1beta </td><td> R &amp; S </td><td> AFL201 </td></tr><tr><td> IL-6 </td><td> CellaGenix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH Farmacia </td><td></td></tr><tr><td> IL-4 </td><td> R &amp; S </td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-alfa </td><td> R &amp; S </td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> BCM CAGT Vector Production Facility </td><td></td></tr><tr><td> Trasferimento al plasma con adattatore femmina luer </td><td> Carta Medical </td><td> 89-550-66J </td></tr><tr><td> Lymphoprep </td><td> Nycomed </td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

Infezione da virus dopo il trapianto di cellule staminali sono tra le cause più comuni di morte, specialmente dopo sangue cordonale (CB) trapianto (CBT) in cui l&#39;organismo di certificazione non contiene numeri notevoli di virus-esperti cellule T in grado di proteggere il destinatario dalle infezioni 1. - 4 Noi e altri hanno dimostrato che virus specifica CTL generati da donatori sieropositivi e infuso al destinatario sono sicuri e protettivi. 5-8 Tuttavia, fino a poco tempo, virus-specifiche cellule T non può essere generato dal sangue del cordone ombelicale, probabilmente a causa del assenza di celle di memoria virus-T specifiche. Nel tentativo di imitare il meglio in condizioni di innesco in vivo di cellule T naive, abbiamo stabilito un metodo utilizzato CB derivate da cellule dendritiche (DC) trasdotte con un vettore adenovirale (Ad5f35pp65) contenente l&#39;antigene immunodominante CMV pp65, quindi guidare specificità delle cellule T verso CMV e adenovirus. 9 Alla iniziazione, usiamo queste macchineDCs strutturati come anche cellule T CB-derivato in presenza delle citochine IL-7, IL-12 e IL-15. 10 Alla stimolazione secondo abbiamo usato EBV-trasformate cellule B, o EBV-LCL, che esprimono sia latente e litiche antigeni EBV. Ad5f35pp65-trasdotte EBV-LCL sono usati per stimolare le cellule T in presenza di IL-15 alla seconda stimolazione. Stimoli successivi utilizzano Ad5f35pp65-trasdotte EBV-LCL e IL-2. Da 50x10 6 cellule mononucleate CB siamo in grado di generare l&#39;alto di 150 x 10 6 cellule T virus-specifici che lisare antigene-pulsate obiettivi e citochine rilascio in risposta alla stimolazione antigenica. 11 Queste cellule sono state prodotte in un modo conforme GMP utilizzando solo la frazione al 20% di una unità di sangue del cordone frazionato e tradotti per l&#39;uso clinico.

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

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

Qui si descrive la prima pratica di fabbricazione (GMP) conforme metodo per produrre virus-specifici linfociti T citotossici (CTL) da sangue del cordone ombelicale, fonte di cellule T prevalentemente ingenuo.

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 ...

Research

JoVE Journal

Immunology and Infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

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Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

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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, ...

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The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...