“Modulazione dell'espressione genica in cellule linfoidi ed epitelioidi infettate con il Virus Erpetico Umano 8 (HHV8) ed analisi dell’attività chemioterapica/antivirale della Distamicina A (DA)”

Abstract

Purpose of this PhD study have been the analysis of the modulation of cellular gene by Human Herpes virus 8 (HHV8) infection and of the potential antiviral effect of Distamycin A (DA), an oligopeptidic antibiotic. This study describes a comprehensive picture of global gene changes soon after HHV-8 active infection of two susceptible cell types. We found that host cell expression changes after only 24 hours of infection, and it is quite different from previous gene induction studies that were carried out at later time points of virus infection. In fact, Poole et al. have used a different primary effusion lymphoma cell line (JSC1) to produce the virus that was used to infect primary endothelial cells for a period of 3–5 weeks, and gene array experiments were done when almost all of the cells changed from typical cobblestone to spindle-shaped morphology and were positive for LANA. Several features were apparent from these analysis: a) there was virtually non-overlapping list of genes in all of two viral subtypes analyzed; b) HHV-8-induced transcriptional profiles in the epithelial and PBMC were closely different. This is probably because the criteria of > 4-fold gene induction as significant is a threshold filter very tight. Nevertheless, the genes that emerge from such comparison will be of greater interest for studying their role in the unique biology of HHV-8. Furthermore, the replicative capacity of the two different strains in the two cell types and the modulation of cell involved in the inflammatory process would seem to indicate that the genotypic difference could be translated into a different virus-host relationship, also attributable to a different pathogenetic mechanism. In addition to identifying specific host response genes within each functional pathway that are already known to be important for HHV-8 infection, our findings identify novel candidate genes that are not yet known in HHV-8 biology. Obtained results show a lack of cytotoxicity of DAlipo on cultured HEK293, RCE and PBMC cells. It might be noted that the CC50 value found in the present study falls at the low extreme of the range of CC50 values reported in the literature. In fact, available data show CC50 values ranging from 35 μM in a Hep-2 cell line to 154 μM in a HeLa cell line and 385 μM in rabbit primary kidney cells. It has been reported that DA inhibits the activity of all DNA polymerases. This mechanism of action could explain its antiviral activity. Because of the lack of information concerning the relevant genetic definition of HHV8 A1 and C3 subtypes from BCBL1 and BC3 cell lines, whether the observed behavior be the result of the expression of specific mutated genes or a non specific consequence of viral strain virulence has to be established. Clearly, further studies are needed to explain the different degree of sensitivity of HHV8 subtypes to DA and Dalipo. The significant antiviral activity of DAlipo and the lack of citotoxicity in epithelial and lymphoid cell lines shown in the present study indicates the compound as an antiherpetic drug potentially useful in both topic and systemic treatments. It could be used either alone or in association with other antiviral treatment, the latter to be employed at lower dosages and for shorter periods of time, thus limiting side effects. The antiviral activity of DAlipo formulation also in lymphoid cell lines may be due to the hydrophobic or amphiphilic drug penetration efficacy increased through inclusion of the drug in liposomes. DAlipo showed, when compared with the free drug, higher antiviral activity against HHV8 A1 and C3 subtypes in PBMCs and might therefore represent a promising DA formulation that could be used also for systemic treatments