Nadal
Fakultäten » Medizinische Fakultät » Kinderspital Zürich: Medizinische Klinik » Infektiologie, Abteilung » Prof. Dr. David Nadal » Nadal
Completed research project
| Title / Titel | Role of the innate immune system in EBV-associated lymphoma development and therapy | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Abstract (PDF, 14 KB) | |||||||||
| Summary / Zusammenfassung | We hypothesize that the innate immunity plays a key role in EBV-dependent tumor induction. This hypothesis is based on enhanced immortalization of human B-cells when triggering TLR9 and decreased expression of the lytic EBV gene BZLF1 coupled with increased expression of the transforming latent EBV gene LMP1. We aim to elucidate the role of TLRs in the propagation of EBV-infected B-cells in vitro and the formation of EBV-harboring tumors in vivo. Thus, we will phenotypically and functionally characterize the EBV-positive Burkitt lymphoma cell line Akata and its EBV-negative clone, the marmoset cell line B95.8, human LCLs as well as human primary B-cells from tonsils for the presence of TLRs and their responsiveness to TLR ligands using in-house quantitative polymerase chain reaction (PCR)-based assays, flowcytometry and enzyme-linked immunosorbent assays. Then, we will assess the effects of selective TLR triggering on the EBV replication phase employing latently EBV-infected cells forced into a lytic phase by immunoglobulin cross-linking or mitogens. Next, we will assess the role of TLR triggering for EBV replication and EBV-associated lymphoma induction in the human-SCID mouse model injected intraperitoneally either with EBV positive or EBV negative Akata cells, or with EBV-infected human primary tonsillar cells. Injection of EBV-infected cells into SCID mice results in lymphoma formation. Dose-response experiments with the most promising TLR ligands will serve to assess their impact on tumor progression and for inducing a switch of latent to lytic infection, respectively. Tumors from sacrificed mice will be investigated by histopathology and for the presence of EBV by immunhistochemistry or in-situ hybridization and propagated in vitro to assess the molecular mechanisms resulting in their malignant outgrowth. We also aim at the molecular characterization of EBV and cellular gene expression in response to TLR signaling using Akata cells and in-house EBV custom oligonucleotide microarray (ODN-chip). Differences in cellular gene expression that could account for the effect observed on EBV activation, tumor formation or tumor progression will be analyzed by whole genome profiling focusing on a) genes differentially regulated in stimulated vs. non stimulated cells; b) genes responding in the same way over time to stimulation; c) gene list mapped to gene ontology. We will confirm these results by quantitative PCR and Western blotting. Finally, we aim to elucidate the nature of signaling pathways involved in modulation of EBV gene expression upon TLR stimulation. We will focus on activation of BZLF1 as readout, characterize domains /sequences of responding promoters, and conduct experiments using episomal reporter plasmids stably transfected into Akata cells. Deletion constructs of the promoter region will be generated and the sequences necessary for activation determined. Introduction of mutations in the binding sequences will be used to analyze their specificity. The nature of the sequences might then be able to reveal which nuclear transcription factor and therefore which signal transduction cascade is likely to be involved in this regulation. We will also functionally investigate key molecules in signal transduction cascades such as kinases by determining their activity through phosphospecific antibodies. | ||||||||
| Keywords / Suchbegriffe | Epstein-Barr virus, innate immunity, lymphoma, transformation, Toll-like receptor, scid mice, gene expression profiling, signaling | ||||||||
| Project leadership and contacts / Projektleitung und Kontakte |
|
||||||||
| Funding source(s) / Unterstützt durch |
Foundation Krebsliga des Kantons ZürichOlga Mayenfisch Stiftung |
||||||||
| Duration of Project / Projektdauer | Dec 2005 to Nov 2008 |