Background: Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal di... More
Background: Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS), as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS1435/1449) and penta-acylated (LPS1690) structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions.
Methodology/principal findings: This study systematically investigated the effects of P. gingivalis LPS1435/1449 and LPS1690 on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs). We found that LPS1435/1449 and LPS1690 differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS1690 but not by LPS1435/1449. In addition, LPS1690 induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS1690 while down-regulated by LPS1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS1690-induced expression of IL-6 and IL-8 in HGFs.
Conclusions/significance: The present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in gingival tissues, thereby contributing to periodontal pathogenesis. Less
Background
Porphyromonas gingivalis lipopolysaccharide (LPS) is a crucial virulence factor strongly associated with chronic periodontitis which is the primary cause of to... More
Background
Porphyromonas gingivalis lipopolysaccharide (LPS) is a crucial virulence factor strongly associated with chronic periodontitis which is the primary cause of tooth loss in adults. It exhibits remarkable heterogeneity containing tetra-(LPS1435/1449) and penta-(LPS1690) acylated lipid A structures. Human gingival fibroblasts (HGFs) as the main resident cells of human gingiva play a key role in regulating matrix metalloproteinases (MMPs) and contribute to periodontal homeostasis. This study investigated the expression and regulation of MMPs1-3 and tissue inhibitors of MMP-1 (TIMP-1) in HGFs in response to P. gingivalis LPS1435/1449 and LPS1690 and hexa-acylated E. coli LPS as a reference. The expression of MMPs 1–3 and TIMP-1 was evaluated by real-time PCR and ELISA.
Results
The MMP-3 mRNA and protein were highly upregulated in P. gingivalis LPS1690- and E. coli LPS-treated cells, whereas no induction was observed in P. gingivalis LPS1435/1449-treated cells. On the contrary, the expression of MMP-1 and −2 was not significantly affected by P. gingivalis LPS lipid A heterogeneity. The TIMP-1 mRNA was upregulated in P. gingivalis LPS1435/1449- and E. coli LPS-treated cells. Next, signal transduction pathways involved in P. gingivalis LPS-induced expression of MMP-3 were examined by blocking assays. Blockage of p38 MAPK and ERK significantly inhibited P. gingivalis LPS1690-induced MMP-3 expression in HGFs.
Conclusion
The present findings suggest that the heterogeneous lipid A structures of P. gingivalis LPS differentially modulate the expression of MMP-3 in HGFs, which may play a role in periodontal pathogenesis. Less
Objectives: The objective of this study was to investigate the effects of dentine primer containing dual antibacterial agents, namely, 12-methacryloyloxydodecylpyridinium... More
Objectives: The objective of this study was to investigate the effects of dentine primer containing dual antibacterial agents, namely, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and nanoparticles of silver (NAg), on dentine bond strength, dental plaque microcosm biofilm response, and fibroblast cytotoxicity for the first time. Methods: Scotchbond Multi-Purpose (SBMP) was used as the parent bonding agent. Four primers were tested: SBMP primer control (referred to as "P"), P+5% MDPB, P+0.05% NAg, and P+5% MDPB+0.05% NAg. Dentine shear bond strengths were measured using extracted human teeth. Biofilms from the mixed saliva of 10 donors were cultured to investigate metabolic activity, colony-forming units (CFU), and lactic acid production. Human fibroblast cytotoxicity of the four primers was tested in vitro. Results: Incorporating MDPB and NAg into primer did not reduce dentine bond strength compared to control (p>0.1). SEM revealed well-bonded adhesive-dentine interfaces with numerous resin tags. MDPB or NAg each greatly reduced biofilm viability and acid production, compared to control. Dual agents MDPB+NAg had a much stronger effect than either agent alone (p<0.05), increasing inhibition zone size and reducing metabolic activity, CFU and lactic acid by an order of magnitude, compared to control. There was no difference in cytotoxicity between commercial control and antibacterial primers (p>0.1). Conclusions: The method of using dual agents MDPB+NAg in the primer yielded potent antibacterial properties. Hence, this method may be promising to combat residual bacteria in tooth cavity and invading bacteria at the margins. The dual agents MDPB+NAg may have wide applicability to other adhesives, composites, sealants and cements to inhibit biofilms and caries. Less
Successful bone tissue engineering depends on the scaffold's ability to allow nutrient diffusion to and waste removal from the regeneration site, as well as provide an ap... More
Successful bone tissue engineering depends on the scaffold's ability to allow nutrient diffusion to and waste removal from the regeneration site, as well as provide an appropriate mechanical environment. Since bone is highly vascularized, scaffolds that provide greater mass transport may support increased bone regeneration. Permeability encompasses the salient features of three-dimensional porous scaffold architecture effects on scaffold mass transport. We hypothesized that higher permeability scaffolds will enhance bone regeneration for a given cell seeding density. We manufactured poly-ɛ-caprolactone scaffolds, designed to have the same internal pore design and either a low permeability (0.688×10(-7)m(4)/N-s) or a high permeability (3.991×10(-7)m(4)/N-s), respectively. Scaffolds were seeded with bone morphogenic protein-7-transduced human gingival fibroblasts and implanted subcutaneously in immune-compromised mice for 4 and 8 weeks. Micro-CT evaluation showed better bone penetration into high permeability scaffolds, with blood vessel infiltration visible at 4 weeks. Compression testing showed that scaffold design had more influence on elastic modulus than time point did and that bone tissue infiltration increased the mechanical properties of the high permeability scaffolds at 8 weeks. These results suggest that for polycaprolactone, a more permeable scaffold with regular architecture is best for in vivo bone regeneration. This finding is an important step toward the end goal of optimizing a scaffold for bone tissue engineering. Less
We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and g... More
We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeast's virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human β-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammatory cytokine secretion (IL-1β and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent. Copyright © 2011 Elsevier Inc. All rights reserved. Less
Inflammation or hypoxia in gingival tissue can induce endoplasmic reticulum (ER) stress, which is related with autophagy. The autophagy is a catabolic process involving t... More
Inflammation or hypoxia in gingival tissue can induce endoplasmic reticulum (ER) stress, which is related with autophagy. The autophagy is a catabolic process involving the degradation of a cell's own components. Although autophagy resulting in the total destruction of the cell is one of cell death types, no conclusive evidence exists for such a process. In order to examine the association of ER stress and autophagy in gingival system, ER stress agents brefeldin A, thapsigargin, and tunicamycin were exposed to human gingival cells. The ER stress agents induced cell death and the expression of ER stress proteins, glucose-regulated protein of 78 kDa (GRP78) and CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP). ER stress also increased the formation of autophagic vesicles with the expression of beclin and LC-3 (microtubule-associated protein1 light chain 3) II, two autophagic markers. ER stress induced the phosphorylation of p38MAPK (mitogen-activated protein kinase), and the p38MPAK inhibitor, SB203580, inhibited the resulting cell death and autophagy. In summary, ER stress induces cell death and autophagy through p38MAPK in human gingival cells. Less
Interleukin-13 receptor-targeted cytotoxin (IL13-PE38) is highly cytotoxic to certain types of human cancers expressing abundant levels of IL-13Ralpha2 chain. Although IL... More
Interleukin-13 receptor-targeted cytotoxin (IL13-PE38) is highly cytotoxic to certain types of human cancers expressing abundant levels of IL-13Ralpha2 chain. Although IL13-PE38 is being tested in a Phase III clinical trial in brain tumors, the activity of IL13-PE38 alone or when combined with taxane, a chemotherapeutic drug for oral squamous cell carcinoma (OSCC), has not been investigated. Here, we show that approximately 40% of OSCCs (n = 50) in a tissue array are strongly positive for IL-13Ralpha2, whereas normal oral mucosa (n = 10) expresses very low or undetectable levels evaluated by immunohistochemistry. IL13-PE38 was highly cytotoxic to OSCC cell lines, but not cytotoxic to normal oral fibroblasts. IL13-PE38 mediated a synergistic antitumor effect with paclitaxel in OSC-19 in vitro and in vivo in the orthotopic OSCC tongue tumor model. Real-time tumor growth was monitored by optical imaging using a Xenogen-IVIS imaging system. Treated animals showed significant (p < 0.05) improvement in survival, which correlated with in vivo imaging of tumor response without evidence of visible toxicity. Gene transfer of IL-13Ralpha2 in oral cancer cells increased sensitivity of OSCC cell line to IL13-PE38 in vitro. Retrovirus-mediated gene-transfer of IL-13Ralpha2 in HSC-3 into tongue tumors in vivo dramatically enhanced the antitumor activity of IL13-PE38, providing complete elimination of established tumors and prolonging survival of these animals. These results indicate that IL13-PE38 in combination with paclitaxel acting via different mechanisms may be a potential treatment option for IL-13Ralpha2 expressing OSCC or for the treatment of non-IL-13Ralpha2 expressing OSCC combined with gene transfer of IL-13Ralpha2. Less
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