The basic structure and biological function of cholera toxin (CT) and heat-labile toxin of enterotoxigenic Escherichia coli (LT), which induces diarrhea in humans, are similar. Both CT and LT act as adjuvants for the enhancement of mucosal and serum antibody responses to the mucosally co-administered protein antigen. CT acts as an adjuvant by inducing IL-4-dependent Th2 cytokine responses, which provide help for antigen-specific secretory IgA (S-IgA) as well as serum IgGl, IgA and IgE antibody responses. On the other hand, LT induces Th1- and partly IL-4-independent Th2-type cells with subsequent serum IgGl, IgG2a and mucosal S-IgA responses. Nontoxic mutant CT (mCT) and the chimeric molecule that combine the A subunit of mCT with the B subunit of LT (mCTA/LTB), like nCT, act as mucosal adjuvants and induce mucosal IgA and systemic IgG and IgA antibody responses. These studies indicate that ADP- ribosyltransferase activity can be separated from the adjuvant properties of CT and LT. Further, those nontoxic enterotoxin derivatives should be considered as candidate mucosal adjuvants for vaccinating humans.

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It is well established that Porphyromonas gingivalis is one of the major pathogens of adult periodontitis. P. gingivalis produces an outer membrane protein with a molecular mass of 40-kDa (40k-OMP). In the present study, in order to assess the potential for application of 40k-OMP in the development of an anti-periodontal disease vaccine, we analyzed 40k-OMP-specific CD4⁺ T helper (Th) cell responses induced in the mucosal as well as systemic compartments when 40k-OMP was administered nasally. When CD4⁺ T cells that were isolated from cervical lymph nodes (CLN) and spleens of mice immunized with 40k-OMP plus cholera toxin (CT) as adjuvant were re-stimulated with 40k-OMP in vitro, significant levels of proliferative responses were induced. In contrast, essentially no increased proliferation occurred in CLN and spleens taken from mice given 40k-OMP alone. Analysis of T helper (Th) 1 (IFN-γ) and Th2 (IL-4, IL-5 and IL-6) cytokine responses showed that 40k-OMP-specific Th cells from both CLN and the spleen produced significant levels of IL-4, IL-5 and IL-6 but did not result in changes in IFN-γ production. In contrast, marginal levels of IL-4, IL-5 and IL-6 production were seen in mice given 40k-OMP alone nasally. These results suggest that nasal administration of 40k-OMP plus CT as an adjuvant can elicit 40k-OMP-specific Th2-type cytokine responses in both mucosal and systemic compartments. Further, the nasal 40k-OMP vaccine possesses the potential for antiperiodontal vaccine.

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Porphyromonas gingivalis has been implicated as a major pathogen in periodontal diseases. The coaggregation factor plays an important role in colonization of P. gingivalis in the subgingival area through aggregation with other extant oral microorganisms. We previously succeeded in gene cloning of the 40-kDa outer membrane protein (OMP) from P. gingivalis and identified the 40-kDa OMP as one of coaggregation factors. The mouse monoclonal antibody Pg-omp-A2, which inhibited the coaggregation activity of P gingivalis, was successfully constructed. In order to develop a useful immunotherapy, it is essential to understand the functional domain expressing aggregation activity. The availability of recombinant 40-kDa OMP and the identification of the antigenic determinant recognized by Pg-omp-A2 will allow for the determination of the functional domain of the coaggregation factor.
In this study, we used a phage-displayed epitope mapping system to examine the functional domain expressing aggregation activity. Peptide-displayed phage clones were isolated by biopanning using Pg-omp-A2 and determined DNA nucleotide sequence and then predicted the amino acid sequence of epitope. The amino acid sequence of epitope region was similar to the region of ⁹⁶IALDQTLGIP¹⁰⁵ in 40-kDa OMP. Next, a series of 6 peptides (designated peptide A to F) in 40-kDa OMP was designed and chemically synthesized, and then we examined their immuno-reactivity by Pg-omp-A2. Pg-omp-A2 recognized the peptide C that contained the IALDQTLGIP amino acid sequence. Furthermore, the peptide C reduced the inhibitory activity of Pg-omp-A2 against the aggregation of P. gingivalis vesicles with Streptococcus gordonii. These findings suggest that the aggregation-associated domain of P gingivalis 40-kDa coaggregation factor may be IALDQTLGIP.

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In order to find functional characteristics of human dental pulp in the irreversible progress of pulpitis, we examined the effects of IL-1β and IL-6 on matrix metalloprotease (MMP) activity in human-dental-pulp derived fibroblastic cells (HDP) compared with human gingival-derived fibroblasts (HGF). The activities of MMPs activated by IL-1β or IL-6 in HDP and HGF were investigated with zymography. MMP-2 activity in conditioned media of both cells cultured with IL-1β or IL-6 were activated, but MMP-9 activity was not activated without plasminogen. When plasminogen was added to the conditioned medium, plasmin and MMP-9 were activated. Plasmin and MMP-9 of HDP-treated IL-6 were more activated than HGF. Next, we examined the effect of these cytokines on the plasminogen activator(PA)/plasmin system. The PA activity in the conditioned medium showed no difference when both cells were incubated with IL-1β. In contrast, when both cells were incubated with IL-6, the PA activity of HDP was higher than that of HGF. By reverse-transcription polymerase chain reaction (RT-PCR), tPA mRNA levels in both cells were enhanced by IL-1β and IL-6, but uPA was not effected. IL-6-stimulated tPA mRNA level of HDP was higher than that of HGF. These findings suggest that IL-6 may play a major role in extracellular matrix (ECM) degradation through stimulation of the PA/plasmin system in HDP of pulpitis.

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Tumor necrotizing factor-alpha (TNF-α) is thought to be an important inflammatory cytokine in the progression of periodontitis. Plasminogen activator (PA) /plasmin cascade plays an important role in the inflammatory process through the activation of metalloprotease. The binding of urokinase-type PA (uPA) to its receptor(uPAR) has been proposed as an important feature of cellular processes requiring extracellular matrix degradation. The uPAR attaches on the cell surface with a glycosylphophatidylinositol (GPI) anchor and serves to localize and accelerate the proteolysis cascade. However, the relation of TNF-α and (PA)/plasmin cascade activation by uPAR in periodontitis is not clear.
In this study, we primary-cultured human-gingival-tissue-derived fibroblasts (hGF) and examined the effect of TNF-α on uPA and uPAR expression in hGF. PA activities in the conditioned medium and cell lysates of hGF were increased by TNF-α treatment. In addition, PA activity released by phosphatidylinositol-specific phospholipase C, which detaches the GPI anchor, was also increased by TNF-α treatment. Furthermore, TNF-α increased the mRNA levels of both uPA and uPAR by reverse transcription-polymerase chain reaction analysis. These findings suggest that the enhancement of uPA and uPAR expressions by TNF-α may play an important role in the progression of periodontitis through pericellular proteolysis.

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Oxygen radicals have been implicated as being involved in cellular damage, oxidation of proteins, membrane lipid peroxidation, inactivation of enzymes, and DNA chain breakage during the processes of aging, inflammation, and injury. Apoptosis is an essential mechanism for the selective elimination of cells that is associated with a variety of disorders and encompasses most cell systems.
We investigated the effects of extracellular oxygen radicals on apoptosis in a cultured human submandibular gland (HSG) cell line and HSG-AZA3 cells, using an acinar cell phenotype that was induced by treatment of HSG cells with 5-azacytidine. The induction of apoptosis was examined after the cells were exposed to an oxygen radical system composed of hypoxanthine (HX) and xanthine oxidase (XOD). The appearance of nuclei with DNA fragmentation was detected by a TUNEL method while the expression of active casepase-3 was found by immunocytochemistry. Apoptosis of HSG and HSG-AZA3 cells induced with HX/XOD was significantly greater, and active caspase-3 staining intensity was greater than in the control.
These findings suggest that caspase-3 expression may be regulated by oxygen radicals and involved in oxygen radical-induced apoptosis of HSG and HSG-AZA3 cells. Moreover, these established cell lines are useful as a model of aging in submandibular glands.

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The objective of the present study is to make a resin by using fluoroalkyl acrylate monomer for the suppression of the adhesion of microbes to dentures surfaces and plaque formation, since it is thought that fluoroalkyl acrylate monomer might develop a denture base material having low affinity for microbes. Here, we could produce polymer beads by adding perfluorooctylethyl acrylate (C8F) to methyl methacrylate (MMA) in order to modify the surface characteristics of the denture base resin, and then investigated microbial adhesion to test specimens.
The water contact angle for C8F-added specimens (C8FA) was higher than that for control specimens (MMA/PMMA), although the surface hardness and tensile strength of the C8FA were slightly lower than those of the control. In addition, the maximum fluorine concentration for the C8FA was at a depth of 0.2 mm. Furthermore, bacterial adhesion to test specimens using Candida albicans was lower for the C8FA, when compared to the control. These findings suggest that the surface characteristics of dentures may be improved by adding a fluoroalkyl acrylate monomer to the filling polymer used in denture base resins.

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The purpose of this study was to compare the production of interleukin-1β (IL-1β) by peptidoglycan from Lactobacillus casei (L. casei) in human pulp-derived fibroblasts from deciduous (DHPF) and permanent teeth (HPF). DHPF and HPF were collected from 6 noncarious deciduous teeth (6-9 year-old individuals) and 6 noncarious permanent teeth (20-25 year old individuals) extracted in the course of orthodontic treatment. Both IL-1β protein production and IL-1β mRNA levels from peptidoglycan- treated DHPF and HPF were clearly enhanced, compared to IL-1β protein production and IL-1β mRNA levels from peptidoglycan-non-treated DHPF and HPF that were not treated by peptidoglycan. Furthermore, IL-1β protein production and IL-1β mRNA levels from peptidoglycan-treated DHPF showed clearly lower levels, compared with IL-1β protein production and IL-1β mRNA levels from peptidoglycan-treated HPF. These findings suggest that pulpitis in deciduous teeth was less induced than pulpitis in permanent teeth, and that L. casei- peptidoglycan stimulated IL-1β production through enhancement of IL-1β mRNA expression from DHPF and HPF.

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Mesenchymal stem cells (MSCs) contain multipotent cells and differentiate into osteoblasts, chondrocytes, and adiphocytes. Recently, MSCs have been used clinically in a tissue-engineering concept, but cell activity varies. It was found that the characteristics of extracellular matrices, type Tcollagen and/or fibronectin matrices, induced the osteoblastic differentiation of MSCs. Six weeks after cells were cultured with type I collagen and fibronectin, they formed mineralized tissue six times higher than other matrices. In this study, using “real time RT-PCR,” we assessed the expression of osteogenesis-related genes (alkaline phosphatase, BMP-2, osteocalcin, and cbfa-1) during osteoblastic differentiation by measuring their mRNA, quantitatively. The expression of the alkaline phosphatase gene increased time-dependently during osteoblastic differentiation until day 12, but expression induced by type I collagen and type I collagen and fibronectin decreased gradually. The expression of osteocalcin and the BMP-2 gene increased time-dependently; osteocalcin gene expression induced by type I collagen and fibronectin was about 6.2 times higher than non-induced cells. Expression of the cbfa-1 gene was different from that of type I collagen, fibronectin, and non-induced cells from day 12 and increased time-dependently. These findings were supported by alkaline phosphatase staining and immunofluoresence with an osteopontin antibody.

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A rare case of an osteosarcoma of the mandible in a 75-year-old person was reported, and the literature on those 75 years and older were reviewed. The clinical, radiological and histopathological findings were similar to those of cases in younger people. In accordance with an increase in the older population, the number of secondary osteosarcomas has increased, but osteosarcoma of the jawbone in those 75 years and older is still very rare.

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The group of enzymes collectively called glucosyltransferases (GTF) plays an important role in dental plaque formation and colonization of cariogenic bacteria on the tooth surface. Streptococcus mutans and Streptococcus sobrinus are predominant mutans streptococci species presented in the human oral cavity. Three kinds of GTFs (GtfB, GtfC, and GtfD) are from S. mutans, and 4 kinds (GTFI, GTFS, GTFT, and GTFU) from S. sobrinus. Recently, the gtfU gene has been molecularly cloned, and nucleotide sequences of the gene were completely analyzed. Thus, all gtf genes are now defined in the DNA database. Detailed information of structure and function of GTFs is important not only in evaluating the nature of virulence but also for the development of a vaccine against dental caries. GTFs have 3 major functional domains, including an N-terminal highly variable region (CD1), a conserved core catalytic region (CD2), and a C-terminal glucan-binding region (GBD). In this study, 3 functional domains in 7 GTFs were compared from motif search results using a protein motif database Pfam. Putative catalytic domains CD1 and CD2 were well maintained in all 7 GTFs while numbers of putative GBD repeat were different in GTFs, and GTFU has a large number of the repeat.

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