Effects of daidzein on alveolar bone loss and internal microstructures of bone in a rat model of experimental periodontitis: a study using micro-computed tomography.

BACKGROUND AND OBJECTIVE: Daidzein is an isoflavone abundant in soybeans, kudzu root and red clover, which have been widely studied for its therapeutic potential. The present study was designed to evaluate the effects of daidzein on alveolar bone loss and internal microstructures of bone in a rat model of experimental periodontitis by assessing morphological data obtained from micro-computed tomography (micro-CT). MATERIAL AND METHODS: Twenty-four male Sprague-Dawley rats were randomly assigned to the following three groups comprising eight animals each: the nonligation (NL) group; the ligation (L) group; and the ligation+daidzein (LD) group. To induce periodontitis, a 4-0 braided silk ligature was tied around the cervical area of the lower-right first molars of rats in groups L and LD. Rats in the LD group were given daily doses of daidzein (10 mg/kg of body weight) by intraperitoneal injection immediately after ligature placement. Two weeks after the placement of ligatures, mandibular block biopsies were scanned using a micro-CT system. RESULTS: Daily administration of daidzein strongly suppressed the ligature-induced loss of alveolar bone height. In addition, when rats were treated with daidzein, the ligature-induced decrease in the bone volume fraction was significantly recovered. Furthermore, daidzein significantly reversed ligature-induced deteriorations in the microarchitecture parameters of trabecular bone, such as trabecular thickness, bone mineral density, trabecular separation and structure model index. CONCLUSION: The study presented here demonstrates, for the first time, that daidzein effectively reduces alveolar bone destruction resulting from experimental periodontitis in rats. Further studies are necessary for the translation of this compound clinically to improve the outcomes of patients diagnosed with periodontitis.

Applications of three-dimensionally scanned models in orthodontics.

The purpose of this study was to investigate clinical applications of the three-dimensional reverse engineering technologies for the analysis of orthodontic models. The measuring accuracy and the process of the 3D model scanning technique were evaluated with respect to linear, surface and volumetric parameters. Orthodontically induced dentoalveolar changes, which have been traditionally evaluated by cephalometric analysis, were assessed by the registration function of Rapidform 2002, a 3D-reverse modeling software in scanned maxillary casts. Three-dimensional digital models are valuable alternatives to conventional casts for model analysis and also yield information which could previously be gathered only by cephalometric superimposition.

Epitope mapping of Porphyromonas gingivalis heat-shock protein and human heat-shock protein in human atherosclerosis.

To identify T- and/or cross-reactive B-cell epitopes of P. gingivalis and human heat-shock protein (HSP)60 in atherosclerosis patients, we synthesized 104 overlapping synthetic peptides spanning whole molecules of P. gingivalis HSP60 and human HSP60, respectively. T-cell epitopes of P. gingivalis HSP were identified with the use of previously established P. gingivalis HSP-reactive T-cell lines. B-cell epitopes of P. gingivalis HSP60 and human HSP60 were identified by the use of patients' sera. Anti-P. gingivalis, anti-P. gingivalis HSP60, or anti-human HSP60 IgG antibody titers were higher in the atherosclerosis patients compared with the healthy subjects. Five immunodominant peptides of P. gingivalis HSP60, identified as T-cell epitopes, were also found to be B-cell epitopes. Moreover, 6 cross-reactive B-cell epitopes of human HSP60 were identified. It was concluded that P. gingivalis HSP60 might be involved in the immunoregulatory process of atherosclerosis, with common T- and/or B-cell epitope specificities and with cross-reactivity with human HSP60.

Establishment of Porphyromonas gingivalis heat-shock-protein-specific T-cell lines from atherosclerosis patients.

Human atherosclerotic plaques contain heat-shock proteins which may serve as potential targets of the immune response in atherosclerosis. Since periodontal infections are suggested as risk factors for the development of cardiovascular diseases, we undertook the present study to evaluate the T-cell immune responses specific to Porphyromonas gingivalis (P. gingivalis) heat-shock protein (hsp)60 in patients suffering from atherosclerosis. Anti-P. gingivalis hsp60 IgG antibody titers were elevated in all patients. We could establish P. gingivalis hsp-specific T-cell lines from the atheroma lesions and the peripheral blood. The T-cell lines were a mixture of CD4+ and CD8+ cells producing the cytokines characteristic of both Th1 and Th2 subsets. The present findings suggest that the T-cell immune response specific to P. gingivalis hsp60 may be involved in the immunopathologic process of atherosclerotic diseases.

Combined periodontal-prosthodontic treatment of early-onset periodontitis--an alternative to implant therapy.

A variety of treatment systems should be available for patients whose dentitions are seriously compromised so that they may select customized treatment modalities that satisfactorily restore occlusal function, consider systemic conditions, and lessen the surgical and financial burdens. These requirements become more demanding when clinicians are faced with advanced cases of rapidly progressive periodontitis. Therefore, it is critical to establish sophisticated multidisciplinary treatment modalities for the successful management of these compromised patients. Obviously, because of various limitations, implant therapy cannot be the only solution. This article reports on the successful long-term management of seriously compromised early-onset periodontitis by a combined periodontal-prosthetic treatment as an alternative to implant therapy.

Lipopolysaccharides from various Capnocytophaga strains possess potent Limulus amoebocyte lysate clotting activity.

Lipopolysaccharides (LPSs) were extracted by the hot phenol-water method from three ATCC strains (C. sputigena ATCC 33612, C. ochracea ATCC 33596, and C. gingivalis ATCC 33624) and three clinical isolates (C. sputigena TE-1, C. ochracea ONO-26, and C. gingivalis M-12). Endospecy was used to determine the Limulus amoebocyte lysate clotting activities. The activities of LPSs from Capnocytophaga strains were stronger than those of Escherichia coli, with the exception of the LPS from C. gingivalis M-12. Except for the LPS from C. sputigena TE-1, the SDS-PAGE analyses of these preparations showed slow-migrating and repeating ladder bands similar to the LPSs of E. coli and Salmonella typhimurium (wild type) and included both the core-lipid A region and various lengths of O-antigen. The LPS from C. sputigena TE-1 possessed fast-migrating bands and did not have an O-side chain.

B-cell mitogenicity and IL-1 beta production of lipopolysaccharides from various Capnocytophaga strains.

To evaluate the etiological roles of Capnocytophaga species in the pathogenesis of periodontal disease, we examined the immunological activities of lipopolysaccharides (LPSs) from various Capnocytophaga strains. All LPSs from various Capnocytophaga species were mitogenic for BALB/c mouse spleen cells, although the responses were lower than those to reference LPSs from Escherichia coli or Salmonella typhimurium. LPSs of C. sputigena strains had polyclonal B cell activation and adjuvant activity and were comparable to reference LPSs. All LPSs from Capnocytophaga strains activated the interleukin-1 beta production from human peripheral monocytes, although the inducing activities of Capnocytophaga LPSs were lower than those of reference LPSs. It appears that LPSs from various Capnocytophaga strains activate certain immunological responses from lymphocytes and monocytes which may be important in the development and pathogenesis of periodontal disease.

Lipid A-associated proteins from Porphyromonas gingivalis stimulate release of nitric oxide by inducing expression of inducible nitric oxide synthase.

BACKGROUND AND OBJECTIVE: The purpose of this study was to examine the effects of lipid A-associated proteins from Porphyromonas gingivalis, a major cause of inflammatory periodontal disease, on the production of nitric oxide and expression of inducible nitric oxide synthase in the murine macrophage cell line, RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. gingivalis lipid A-associated protein-induced nitric oxide production. MATERIAL AND METHODS: The lipid A-associated proteins from P. gingivalis 381 were prepared by standard hot phenol-water extraction of endotoxin isolated by the butanol method. Nitric oxide production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of inducible nitric oxide synthase and analysis of reverse transcription-polymerase chain reaction products were carried out. RESULTS: We found that P. gingivalis lipid A-associated proteins can induce inducible nitric oxide synthase expression and stimulate the release of nitric oxide without additional stimuli, and we demonstrated that multiple signaling pathways, such as nuclear factor-kappaB, microtubule polymerization, protein tyrosine kinase, protein kinase C, and mitogen-activated protein kinase cascades, are involved in P. gingivalis lipid A-associated protein-stimulated nitric oxide production. The production of nitric oxide required l-arginine. CONCLUSION: The present study clearly shows that P. gingivalis lipid A-associated proteins fully induced inducible nitric oxide synthase expression and nitric oxide production in RAW264.7 cells in the absence of other stimuli. The ability of P. gingivalis lipid A-associated proteins to promote the production of nitric oxide may be important in the pathogenesis of inflammatory periodontal disease.

Recognition and phagocytosis of multiple periodontopathogenic bacteria by anti-Porphyromonas gingivalis heat-shock protein 60 antisera.

The present study has been performed to evaluate Porphyromonas gingivalis heat shock protein (HSP) 60 as a candidate vaccine to protect against multiple putative periodontopathic bacteria. Mouse anti-P. gingivalis HSP antisera demonstrated the elevated IgG antibody titers against the multiple bacteria tested and cross-reacted with heat-induced bacterial proteins of the target bacteria. The antisera also demonstrated a significantly higher opsonophagocytosis function against all the target bacteria than the control sera (P<0.01). We concluded that P. gingivalis HSP 60 could potentially be developed as a vaccine against multiple periodontopathic bacteria.

Production of microbial polyester by fermentation of recombinant microorganisms.

Polyhydroxyalkanoates (PHAs) can be produced from renewable sources and are biodegradable with similar material properties and processibility to conventional plastic materials. With recent advances in our understanding of the biochemistry and genetics of PHA biosynthesis and cloning of the PHA biosynthesis genes from a number of different bacteria, many different recombinant bacteria have been developed to improve PHA production for commercial applications. For enhancing PHA synthetic capacity, homologous or heterologous expression of the PHA biosynthetic enzymes has been attempted. Several genes that allow utilization of various substrates were transformed into PHA producers, or non-PHA producers utilizing inexpensive carbon substrate were transformed with the PHA biosynthesis genes. Novel PHAs have been synthesized by introducing a new PHA biosynthesis pathway or a new PHA synthase gene. In this article, recent advances in the production of PHA by recombinant bacteria are described.

High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli.

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.

Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of Poly(3-hydroxybutyrate) in Escherichia coli.

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, beta-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.

Polarization of Porphyromonas gingivalis-specific helper T-cell subsets by prior immunization with Fusobacterium nucleatum.

Antigen-specific T-cell clones were obtained from mice immunized with Fusobacterium nucleatum ATCC 10953 and/or Porphyromonas gingivalis 381. 10 BALB/c mice per group were immunized with F. nucleatum followed by P. gingivalis, or with P. gingivalis alone by intraperitoneal injection of viable microorganisms. Spleen T cells were isolated and stimulated in vitro with viable P. gingivalis cells to establish P. gingivalis-specific T-cell clones. T-cell phenotypes and cytokine profiles were determined along with T-cell responsiveness to F. nucleatum or P. gingivalis. Serum immunoglobulin G antibody titers to F. nucleatum or P. gingivalis were also determined by enzyme-linked immunosorbent assay. All the T-cell clones derived from mice immunized with F. nucleatum followed by P. gingivalis demonstrated Th2 subsets, while those from mice immunized with P. gingivalis alone demonstrated Th1 subsets based on the flow cytometric analysis and cytokine profiles. All T-cell clones from both groups were cross-reactive to both P. gingivalis and F. nucleatum antigens. Phenotypes of T-cell clones were all positive for CD4. Mean post-immune serum IgG antibody levels to F. nucleatum or P. gingivalis were significantly higher than the pre-immune levels (P < 0.05, P < 0.01, respectively). There were no significant differences in the antibody titers between the two groups. It was concluded that P. gingivalis-specific T cells initially primed by cross-reactive F. nucleatum antigens were polarized to Th2 subset, while T cells stimulated with P. gingivalis alone maintained the profile of Th1 subset.

Isolation, expression, and nucleotide sequence of the sod gene from Porphyromonas gingivalis.

The sod gene coding for the Mn/Fe-dependent superoxide dismutase (SOD) enzyme has been isolated on a 5.9-kb DNA fragment from Porphyromonas gingivalis ATCC 53977. SOD activity can be expressed from the P. gingivalis fragment and from a subcloned fragment in Escherichia coli. However, the enzyme does not appear to be expressed from its own promoter in E. coli cells. The nucleotide sequence of the gene has been determined, and the deduced amino acid sequence of the enzyme is nearly identical to that of the enzyme purified from P. gingivalis 381 and shares extensive sequence similarity with comparable enzymes from E. coli.

Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis.

The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP).

Clinical, microbiological and immunological studies on recurrent periodontal disease.

The present study was performed to evaluate the clinical, microbiological and immunological aspects in the early stages of recurrent periodontal disease. After clinical monitoring of pockets with recent evidence of disease recurrence, microbiological samples for cultural analysis, serum and gingival crevicular fluid (GCF) samples were taken for IgG antibody analysis from 14 sites, 7 in 6 recurrent periodontitis patients and 7 in 7 periodontally healthy control subjects. IgG responses of serum antibody to 8 gram-negative bacterial strains were compared with those of GCF sampled from the recurrent site. The results clearly demonstrated the predominance of Bacteroides gingivalis in most subgingival plaque samples during the early stages of disease recurrence; the mean proportions of B. gingivalis were significantly different from those of the healthy sites (p less than 0.05). 4 out of 6 serum samples showed the elevated antibody responses to B. gingivalis 381; and this was closely correlated to homologous infection by this micro-organism in recurrent sites. Elevated serum antibody responses were also noted to Eikenella corrodens 1073 and Actinobacillus actinomycetemcomitans Y4. However, no relationship with homologous infection was found for A. actinomycetemcomitans. 3 out of 6 GCF samples had greater antibody titers than the serum, suggesting local antibody synthesis by the gingival cells in the recurrent pockets. The present study showed that B. gingivalis might play an important role in the pathogenesis of disease recurrence in its early stages.

Capsular polysaccharide-fimbrial protein conjugate vaccine protects against Porphyromonas gingivalis infection in SCID mice reconstituted with human peripheral blood lymphocytes.

The effect of immunization with either a Porphyromonas gingivalis fimbrial protein, a capsular polysaccharide, or a capsular polysaccharide-fimbrial protein conjugate vaccine were compared in hu-PBL-SCID mice. A significantly higher human immunoglobulin G antibody response and the highest degree of in vivo protection against bacterial challenge was observed in the group immunized with the conjugate vaccine. It was concluded that capsular polysaccharide-fimbrial protein conjugate from P. gingivalis could potentially be developed as a vaccine against periodontal infection by P. gingivalis.

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.

Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation.

High-cell-density fed-batch cultures of Pseudomonas putida were carried out for the production of medium-chain-length polyhydroxyalkanoates (PHAs) using oleic acid as a carbon source. By employing an optimal feeding strategy without the limitation of any nutrient, a high cell concentration of 173 g/L was achieved, but the PHA concentration and PHA content were only 32.3 g/L and 18.7 wt%, respectively. To increase the PHA concentration and content, phosphorus limitation was applied during fed-bath culture by reducing the initial KH(2)PO(4) concentration. When the initial KH(2)PO(4) concentration was reduced to 4 g/L, cell concentration, PHA concentration, and PHA content obtained in 38 h were 141 g/L, 72. 6 g/L, and 51.4 wt%, respectively, resulting in a high productivity of 1.91 g PHA/L per hour.

Identification of T-cell epitopes of Porphyromonas gingivalis heat-shock-protein 60 in periodontitis.

The heat shock proteins (hsp) of bacterial species are considered to be involved in regulating the autoimmune mechanism in human diseases due to the considerable homology of their sequences with human hsp. To elucidate how stress proteins contribute to the immunopathogenesis of periodontitis, mononuclear cells from gingival connective tissue of 10 periodontitis patients were simulated with Porphyromonas gingivalis hsp60. T-cell lines reactive to P. gingivalis hsp60 were established from each patient to define T-cell epitope specificities. Anti-P. gingivalis IgG antibody titres were elevated in all patients. We could establish P. gingivalis hsp-reactive T-cell lines from gingival mononuclear cells that were mixtures of CD4+ and CD8+ cells. Of 108 overlapping synthetic peptides spanning the whole P. gingivalis hsp60 molecule, 10 peptides with epitope specificities for T-cells were identified, and were identical to those reported be B-cell epitopes in periodontitis.