HUMAN HERPESVIRUS 8 UPREGULATES ACTIVATING TRANSCRIPTION FACTOR 4 (ATF4) and REAL TIME PCR TO ASSESS TOTAL BACTERIAL LOAD IN CHRONIC WOUNDS.

I. Human Herpesvirus 8 Upregulates Activator Transcription Factor 4 BACKGROUND: Human herpesvirus 8 (HHV-8) is the primary etiologic agent of Kaposi’s sarcoma, a highly vascularised neoplasm of endothelial origin characterized by inflammation, neoangiogenesis, and by the presence of characteristic spindle cells. HHV-8 angiogenic activity is due to the activation of NF-kB and the subsequent induction of MCP-1 synthesis. MCP-1 is a chemokine produced by macrophages and endothelial cells in response to different stimuli, and is a direct mediator of angiogenesis. HHV-8-induced angiogenesis is MCP-1 dependent. However, HHV-8 activation of MCP-1 is not completely dependent on NF-kB induction and another cellular factor is involved. A potential candidate is the cellular activating transcription factor 4 (ATF4), a stress responsive gene. ATF4 is upregulated in several condition, including ER-stress, viral infection (e.g. CMV) and in tumours. AIM: The aim of the research is to study the interaction between HHV-8 and ATF4, and verify whether ATF4 is involved in angiogenesis characteristic of HHV-8 infection. METHODS: To demonstrate the effect of HHV-8 on ATF4 expression, Jurkat cells were infected with a cell-free viral inoculum, obtained in our laboratory by stimulating reactivation of latent HHV-8 in chronically infected cells (PEL-derived). HHV-8 reactivation from latency was assessed by transfection of BC-3 and BCBL-1 cells (PEL-derived) with pCG-ATF4 recombinant plasmid. DNA or RNA were analysed by PCR, rtPCR or quantitative real time PCR. Promoters activation was assessed by luciferase assays. RESULTS: HHV-8 upregulates the expression of ATF4 gene, and overexpression of ATF4 is able to increase replication and transcription of HHV-8, but not to reactivate HHV-8 from latency. Preliminary results show that ATF4 activates the MCP-1 promoter in absence of the NF-kB binding sites. CONCLUSION: HHV-8 induces ATF4 expression during the productive infection, probably to obtain advantage for its replication and neoplastic development. ATF4 might be implicated in HHV-8 induced tumorigenesis, being involved in several aspects of viral replication, and could represent a potential therapeutic target for HHV-8 induced transformation. II. Real Time PCR To Assess Total Bacterial Load in Chronic Wounds BACKGROUND: Wounds and wound healing are important issues in chronic patients (i.e. diabetes, pressure). Critical colonization and infection are strictly linked to a delay in wound healing. Analysis of pathogens present in chronic wound is an essential aspect for the wound care. Classic microbiologic methods have several limits to ensure the correct analysis of ulcer environment. AIM: To analyse total bacterial load by a single quantitative real time PCR reaction in swabs and biopsies obtained from infected chronic wounds treated with an innovative hydrophobic dressing. METHODS: Biopsies were collected at the beginning and after 4 weeks of treatment, and swabs were collected once a week for 4 weeks. Real time PCR was carried out on DNA extracted from biopsies and swabs amplifying a region of the 16S rRNA gene, highly conserved among bacteria. Moreover, DNA extracted from biopsies was also analysed for the detection of 2 anaerobic bacteria (B. Fragilis, F. necrophorum, frequently associated with delayed healing) by real time PCR amplifying specific unique regions of their genome. In parallel, classical culturing methods were performed on biopsies searching for Staphylococcus and Pseudomonas species. RESULTS: We evaluated the correlation between the molecular data obtained by real time PCR and the clinical data, in particular considering the area of the wound. We observed a mean 253-fold decrease of the total bacterial load in 10/20 wounds those also showed an average 58% decrease of their area. This 10 wounds showed a positive correlation between clinical and molecular data. In 5/20 wound, we found a non significant 5,2-fold decrease of the total bacterial load, correlate with a 27% increase of the wound’s area. Thus, 75% of molecular results (15/20 wounds) were correlate to the clinical data. In contrast, classical culturing method did not correlate with the clinical data, confirming that classical methods have several limits and disadvantages. B. Fragilis was present in 10/20 wounds, and F. necrophorum in 2/20 wounds. CONCLUSION: The molecular approach can be considered a reliable and rapid test to assess infection levels in chronic wounds, being more sensitive than the classic cultural techniques. The research of specific pathogens is not sufficient to assess the outcome of the wounds, whereas total bacterial load can give a prognostic value to the wound care.