Viral-bacterial interactions in the pathogenesis of human endodontic disease
Creators: name: Gu, Fang
Publisher: name: University of North Carolina at Chapel Hill
Year: 2010

description: 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 also significantly higher in the presence of EBV. The result from our in vivo study showed that a greater than 5-fold increase was detected in bacterial DNA levels from pulp tissue of symptomatic patients compared to controls (P=0.004). These differences were not detected between asymptomatic patients and the control (P= 0.275). Likewise, total bacterial activity (RNA) was 5-fold higher in symptomatic patients than the asymptomatic (P=0.027). Lactobacillus sp., Fusobacterium sp. and Actinomyces sp. were present in inflamed pulp tissue, but not in healthy pulp tissue. Among the samples with Streptococcus sp., asymptomatic pulp tissues had 30 fold higher levels, and symptomatic pulp tissues had 5 folds higher levels detected compared to controls (P=0.001, P=0.003 respectively). Interestingly, while EBV was widely detected, CMV was detected only in periapical tissues. Greater than a 30-fold increase in EBV DNA was detected in pulp tissue from symptomatic individuals compared to controls (P<0.001). Further, a 30-fold difference was shown in EBV detection in pulp tissue from symptomatic compared to asymptomatic (P=0.028) patients. The quantity of EBV in the pulp tissue from asymptomatic patients was comparable to the control (P=0.403). Real-time RT-PCR analysis of inflammatory gene expression in pulp tissues detected expression in disease but not within healthy controls. The IL-6 gene was expressed at levels 11-fold higher in symptomatic pulp than in asymptomatic pulp (P=0.026). Regression analysis indicated that inflammatory gene expression levels were associated with bacterial transcription and with EBV viral load. Collectively, our in vitro results show that E. faecalis and EBV may have synergistic effect during inflammatory process. Our in vivo results suggest a significant association between bacteria, EBV and inflammation in symptomatic disease within the pulp tissue. CMV is more tightly associated with chronic infection than acute infection. These results support a paradigm shift in which elevated bacterial/viral replication and inflammation are associated with endodontic infection.