AbstractsBiology & Animal Science

In this study, we hypothesized that herpes viruses, EBV and/or HCMV contribute to endodontic disease via herpes virus-bacterium-host response interactions. In our in vitro study, we investigated potential pathogenic interactions between Epstein-Barr virus (EBV) and the Gram-positive bacterium, Enterococcus faecalis. In our in vivo study, we hypothesized that individuals with symptomatic endodontic disease would have elevated bacterial/viral replication and inflammation compared to that of asymptomatic individuals. In our in vitro study, EBV reactivation within latently infected cells was assessed following exposure to E. faecalis metabolic end products and cell wall by quantitative PCR of media for EBV virions and of cellular mRNA for viral gene expression. To determine potential mechanisms of EBV reactivation, the effect of E. faecalis mediated activation of latent EBV was evaluated when the cells were simultaneously exposed to bacterial products and specific pharmacologic inhibitors of signal transduction pathways. The growth and virulent gene expression of E. faecalis when exposed to lymphoid cells with and without latent EBV infection were evaluated using real-time PCR. To assess the role of viral and bacterial interaction on the host response, transcription of cellular inflammatory genes was determined using real-time RT-PCR. In our in vivo study, twenty pulp tissue samples were collected from patients diagnosed with irreversible pulpits, 10 with symptoms of self-reported severe pain (>7 on 10-point pain scale, Symptomatic) and 10 without self-reported pain (Asymptomatic), respectively. As controls, ten pulp tissue samples were collected from extracted healthy 3rd molars, which had no fracture, caries, or periodontal disease. Additionally, two periapical tissue samples were collected during apicoectomy from patients with persistent periapical infection after root canal treatment. Total DNA and RNA were extracted from pulp tissue using DNeasy and RNeasy kit (Qiagen), respectively. Quantification of total bacteria, Streptococcus sp., Lactobacillus sp., Fusobacterium sp., Actinomyces sp., E. faecalis, EBV and Human Cytomegalovirus (HCMV) were assessed using quantitative PCR (qPCR) for consensus or organism specific DNA. Total bacterial RNA was used to indicate transcriptional activity and was evaluated using quantitative reverse-transcriptase PCR (RT-PCR). The expression of inflammatory genes including IL-6, IL-10, TNF-[alpha], TNF-[beta] and IFN-[beta] was evaluated using quantitative reverse-transcriptase PCR. The result from our in vitro study showed that lipoteichoic acid (LTA), a cell wall component of E. faecalis, can re-activate latent EBV. E. faecalis LTA -mediated induction of lytic EBV infection was significantly reduced by both a TLR2 antagonist and an inhibitor of the NF-kB/Ikb pathway. Interestingly, the growth of E. faecalis increased 5-fold with presence of EBV. The expression of E. faecalis virulence genes were significantly increased in presence of EBV. Proinflammatory cytokine expression was…