An In Vitro Model of Endemic Burkitt’s Lymphoma (eBL) Pathogenesis; Cooperation of Plasmodium Falciparum and Epstein Barr Virus in DNA Damage Mediated Via Activation Induced Cytidine Deaminase.

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Plasmodium falciparum (P. falciparum) and Epstein-Barr virus (EBV) infections are contributors in the pathogenesis of endemic Burkitt’s lymphoma (eBL), although the precise mechanism of their synergy remains elusive. Reports suggest that the role of P. falciparum is indirect, creating a permissive environment for the outgrowth of EBV. EBV on the other hand invades and immortalizes lymphocytes in vitro and upregulates activation-induced cytidine deaminase (AID), a DNA repair enzyme responsible for diversifying the antibody repertoire and a potent mutagen capable of inducing the genetic damage characteristic of eBL. It is yet to be shown how exposure to P. falciparum affects the expression of AID in lymphocytes. The aim of this work was to investigate the possible direct role of P. falciparum in eBL lymphomagenesis by exploring parasite-lymphocyte interactions and AID expression after exposure to P. falciparum and/or EBV. Malaria positive slides were examined for parasite-lymphocyte interactions and primary tonsillar mononuclear cells (MNCs) were co-cultured with RBCs infected with up to 5% parasitemia of the 3D7 strain of P. falciparum. Geimsa stained thin smears were made from these co-cultures and examined for parasite-MNC interactions over a five day period. No direct parasite-MNC interaction was observed from all slides examined. The levels of AID mRNA in MNCs was measured by qPCR after in vitro exposure to P. falciparum and/or EBV, and in the presence or absence of 2µg/ml cyclosporine. P. falciparum induced up to a 6-fold increase in AID over unstimulated controls, EBV induced a 13-fold maximum increase, and both pathogens together induced up to a 22-fold increase in AID. With cyclosporine, AID mRNA levels in the P. falciparum stimulated cultures remained unchanged. EBV alone induced a 22- fold increase in AID and both pathogens together induced a 42-fold increase in AID. DNA damage was estimated by Comet Assay and quantified with an algorithm from the Comet Assay Project Lab (CASP). DNA comets revealed that P. falciparum induced moderate DNA damage in MNCs with up to 5.6% and 10% DNA in tails of comets with and without cyclosporine respectively. Cultures stimulated with EBV recorded DNA damage of up to 16% and 13% DNA in tails of comets with and without cyclosporine respectively; and both pathogens induced DNA damage with up to 11% and 16% DNA in tails of comets with and without cyclosporine respectively. The levels of DNA damage in these cells correlated with AID levels and demonstrate that P. falciparum plays a direct role in eBL pathogenesis, by inducing AID expression to levels similar to that expressed in BL cells and cooperating with EBV to induce abnormally high levels of AID and DNA damage.
A Thesis submitted to the School of Graduate Studies, Kwame Nkrumah University of Science and Technology, Kumasi, in partial fulfilment of the requirements for the Degree of Master of Philosophy, June-2012