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Title: Evaluation of the role of Epstein-Barr virus and cellular gene expression in paediatric and adult transplant recipients
Author: Auburn, Helen
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2012
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Post-transplant lymphoproliferative disease (PTLD) is a life-threatening complication after solid organ, bone-marrow and haematopoietic stem cell transplantation. While early diagnosis is known to predict encouraging outcomes, no specific markers exist for the early detection of PTLD. Epstein-Barr virus (EBV) infection is considered to be an important risk factor for PTLD. However, the correlations between EBV DNA loads and the onset of PTLD are inconclusive. The aim of this study was to identify potential biomarkers for the early onset of PTLD. To accomplish this, we examined EBV and cellular gene expression patterns in (i) the context of EBV lytic induction in a lymphoblastoid cell line (LCL) model system, (ii) PTLD versus non-PTLD transplant patients. In addition we examined the contributions of lytic virus replication vs. latent infection to the onset of PTLD. We used real-time two-step RT-PCR assays with SYBR green detection to analyse EBV gene expression (76 lytic, 12 latent [n = 88 genes]), following 12-O-tetradecanoyl-13-phorbol acetate/sodium butyrate (TPA/NaB) induction of Raji cells. We identified 22 highly induced EBV genes (>90-fold) at 24 hrs post-induction. Using genome-wide Affymetrix microarrays, we identified cellular genes that were highly differentially expressed and regulated during the EBV lytic induction phase, 112 of which were regulated specifically by EBV, and 14 chosen for further study. We developed and validated real-time two-step TaqMan RT-PCR assays for clinical validation. Altogether, we evaluated 17 cellular and 14 EBV candidate genes in whole blood from paediatric solid organ transplant (SOT) recipients and adult umbilical cord transplant (UBCT) recipients, with and without PTLD. We detected a higher number and level of expression of EBV latent and lytic genes in PTLD patients, notably, BALF5, EBNA-LP and LMP-1, and in association with viral loads. Further, we detected latency III and more varied EBV latent gene expression patterns in PTLD. We also detected four cellular genes (CXCL9, CXCL10, CDC2, and CHI3L1) that were differentially expressed in PTLD vs. non-PTLD patients. To conclude, these findings suggest a basis for the likely microenvironment in which PTLD could develop. Further evaluation of candidate EBV and cellular markers could facilitate a more specific diagnosis of PTLD.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available