Bactericidal Effect of Triple Antibiotic Paste against Enterococcus faecalis in Dentinal Tubules: An Ex Vivo Study.

OBJECTIVE: The aim of this study was to investigate the bactericidal effect of various concentrations of triple antibiotic paste (TAP) against Enterococcus faecalis (E. faecalis) in dentinal tubules using a bacterial culture assay and confocal laser scanning microscope (CLSM). METHODS: Ninety human teeth were contaminated with E. faecalis (ATCC 29212) and randomly allocated into 5 groups; the negative control (without TAP), 1 mg/ml, 5 mg/ml, 7.5 mg/ml and 10 mg/ml TAP (n=18). After a 3-week TAP treatment, samples were collected from the root canal space, root dentin at 100-µm and 200-µm depth. The collected samples were subjected to a bacterial culture assay (n=10). Eight roots from each group underwent CLSM analysis to determine the live/dead bacterial cells. RESULTS: The bacterial culture assay results indicated that the negative control samples were all culturable. The number of culture-positive samples decreased after TAP treatment at 1, 5, 7.5 and 10 mg/ml, with 2, 2, 1 and 0 culturable samples, respectively. However, there was no significant difference among the TAP treatments. Surprisingly, the CLSM analysis demonstrated live bacteria in the dentinal tubules in all samples. The negative control had 52.36%+-3.24 live bacteria. TAP treatment at 10 mg/ml had the lowest percentage of live bacterial cells (40.58%+-5.40), followed by 7.5 mg/ml (44.14%+-6.03), 5 mg/ml (46.31%+-5.32) and 1 mg/ml (52.55%+-8.82). The percentage of live cells in the 10 mg/ml, 7.5 mg/ml and 5 mg/ml TAP groups were significantly lower than the 1 mg/ml TAP and negative control groups. CONCLUSION: TAP treatment significantly decreased the percentage of viable E. faecalis cells in the dentinal tubules and its bactericidal effect was dose-dependent.

In vitro bactericidal efficacy of a new triple antibiotic paste formulation against Enterococcus faecalis biofilm.

The aim of this study was to evaluate the bactericidal efficacy on Enterococcus faecalis biofilm of a novel, low-concentration triple-antibiotic paste (TAP-L2) in propylene glycol/carboxymethylcellulose vehicle (VEH-2), a ready-to-use prototype, compared with two currently used products; a low-concentration (TAP-L1) and a high-concentration paste (TAP-H1) mixed in macrogol/propylene glycol vehicle (VEH-1). Sixty-two root canals were infected with E. faecalis biofilm, medicated with (a) TAP-L1, (b) TAP-L2, (c) TAP-H1, (d) VEH-1 or (e) VEH-2 (n = 10) and incubated for 21 days. Live/dead assays were performed using confocal laser scanning microscopy (CLSM), and dead bacteria (%) were calculated. The TAP-H1 group demonstrated the highest dead bacteria (67.54 ± 2.38%), which was significantly higher than the TAP-L2 and TAP-L1 groups (56.85 ± 7.11% and 54.23 ± 10.19%) (p < 0.05). The TAP groups demonstrated significantly higher dead bacteria than the VEH-1 and VEH-2 groups (47.51 ± 6.41% and 45.14 ± 8.28%) (p < 0.05). The ready-to-use TAP-L2 had antibacterial activity comparable to TAP-L1, both of which were lower than TAP-H1.

Post-translational control of IL-1ß via the human papillomavirus type 16 E6 oncoprotein: a novel mechanism of innate immune escape mediated by the E3-ubiquitin ligase E6-AP and p53.

Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1ß) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1ß production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1ß processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1ß regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1ß was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1ß that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1ß is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1ß levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1ß regulation which ultimately inhibits the secretion of IL-1ß in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1ß towards cervical cancer could be discerned. Hence, attenuation of IL-1ß by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy.

HPV16 oncoproteins promote cervical cancer invasiveness by upregulating specific matrix metalloproteinases.

Production of matrix metalloproteinases (MMPs) for degradation of extracellular matrix is a vital step in cancer metastasis. We investigated the effects of HPV16 oncoproteins (16E6, 16E6*I and 16E7), either individually or combined, on the transcription of 7 MMPs implicated in cervical cancer invasiveness. The levels of 7 MMPs reported to be increased in cervical cancer were determined in C33A stably expressing different HPV16 oncoproteins using quantitative RT-PCR and compared with invasion ability of cell lines using in vitro invasion and wound healing assays. Overexpression of MMP-2 and MT1-MMP was detected in HPV16E6E7 expressing cells which correlated with increased cell invasion. Combination of HPV oncoproteins always showed greater effects than its individual form. Inhibition of cell invasion using a specific MMP-2 inhibitor, OA-Hy, and anti-MT1-MMP antibody confirmed that invasion in these cells was dependent on both MMP-2 and MT1-MMP expression. Depletion of HPV16E6E7 by shRNA-mediated knock-down experiments resulted in decreased MMP-2 and MT1-MMP expression levels as well as reduced invasion ability which strongly suggested specific effects of HPV oncoproteins on both MMPs and on cell invasion. Immunohistochemistry study in invasive cervical cancers confirmed the enhanced in vivo expression of these two MMPs in HPV16-infected cells. In addition, possible sites required by HPV16E6E7 on the MMP-2 and MT1-MMP promoters were investigated and PEA3 (at -552/-540 for MMP-2, -303 for MT1-MMP) and Sp1 (at -91 for MMP-2, -102 for MT1-MMP) binding sites were shown to be essential for mediating their transactivation activity. In conclusion, our study demonstrated that HPV16E6 and E7 oncoproteins cooperate in promoting cervical cancer invasiveness by specifically upregulating MMP-2 and MT1-MMP transcription in a similar manner.

Transactivation activity of human papillomavirus type 16 E6*I on aldo-keto reductase genes enhances chemoresistance in cervical cancer cells.

The oncogenic E6 proteins produced by high-risk human papillomaviruses (HPVs) are invariably expressed in cervical carcinomas and are multifunctional proteins capable of affecting host-cell proliferation by binding and deregulating key host molecules such as p53. High-risk HPVs, including HPV16, have the unique ability to splice the E6 viral transcript, resulting in the production of a truncated E6 protein known as E6*I whose precise biological function is unclear. This study explored the changes in gene expression of the cervical cancer C33A cell line stably expressing HPV16 E6*I (16E6*I) and observed the upregulation of ten genes. Two of these genes were aldo-keto reductases (AKR1Cs), AKR1C1 and AKR1C3, which have been implicated in drug resistance. The results demonstrated that expression of 16E6*I, but not full-length E6, specifically increased AKR1C1 transcript levels although it did not alter AKR1C2 transcript levels. HPV16 E7 alone also had the ability to cause a moderate increase in AKR1C3 at both mRNA and protein levels. Site-directed mutagenesis of 16E6*I revealed that transactivation activity was abolished in R8A, R10A and T17A 16E6*I mutants without altering their intracellular localization patterns. Loss of transactivation activity of the 16E6*I mutants resulted in a significant loss of AKR1C expression and a decrease in drug resistance. Analysis of the AKR1C1 promoter revealed that, unlike the E6 protein, 16E6*I does not mediate transactivation activity solely through Sp1-binding sites. Taken together, it was concluded that 16E6*I has a novel function in upregulating expression of AKR1C and, in concert with E7, has implications for drug treatment in HPV-mediated cervical cancer.