Effects of new over-the-counter periodontal ointment-containing applicator with single-tuft brush on cytokine levels in gingival crevicular fluid during supportive periodontal therapy phase: a randomized double-blind clinical trial.

BACKGROUND AND OBJECTIVE: The biochemical effects of an over-the-counter (OTC) medication were studied, which consists of a single-tuft brush containing cetylpyridinium chloride as a bactericidal agent, dipotassium glycyrrhizate as an anti-inflammatory drug and allantoin as a promoter of cell proliferation and wound healing, for delivery to hardly brushed sites. MATERIAL AND METHODS: This randomized controlled double-blind study was performed in 61 subjects with chronic periodontitis in supportive periodontal therapy phase (test group: n = 27; placebo group: n = 28; dropout: n = 6). The OTC medication was self-applied twice a day for 12 wk to two molars with probing pocket depths of 4-6 mm. Biochemical indicators were evaluated at baseline and 12 wk using the suspension array system for eight cytokines and chemokines (interleukin [IL]-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-10, monocyte chemoattractant protein-1 and tumor necrosis factor [TNF]-α) in gingival crevicular fluid. RESULTS: The levels of IL-1ß, IL-6, IL-8 and TNF-α remained significantly lower in the test group compared to the placebo group. In the placebo group, when the probing pocket depth at baseline was 4 mm, IL-1ß increased, particularly in the second molar tooth, and the greatest increase was seen when PPD at baseline was 5-6 mm. In the test group, IL-1ß decreased markedly in cases with furcation involvement and low bleeding on probing at baseline. In both groups, IL-1ß, IL-6 and TNF-α were closely correlated with each other. CONCLUSION: This OTC medication is biochemically effective for steady chronic periodontitis in the supportive periodontal therapy phase.

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes.

BACKGROUND AND OBJECTIVE: Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. MATERIAL AND METHODS: To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. RESULTS: Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. CONCLUSION: We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression.

Overexpression of Smad2 inhibits proliferation of gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Spatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor ß (TGF-ß) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-ß, has an inhibitory effect on re-epithelialization during gingival wound healing. Therefore, we investigated the effects on migration and proliferation status, and intra/extracellular signaling regulated by Smad2 overexpression in gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells were isolated from the palatal gingival tissue of transgenic mice overexpressing Smad2 driven by the Keratin14 promoter. Smad2 expression was identified by western blotting and immunofluorescence analysis. Scratch assay and 5-bromo-2'-deoxyuridine staining were performed to assess cell migration and proliferation. To inactivate TGF-ß type I receptor, the cultures were supplemented with SB431542. Secreted TGF-ß was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium. RESULTS: Smad2-overexpressing cells were confirmed to have significant phosphorylated Smad2 in the nucleus. Scratch assay and 5-bromo-2'-deoxyuridine staining indicated that Smad2-overexpressing cells showed no significant differences in migration, but had reduced proliferation rates compared to wild-type controls. SB431542 significantly inhibited Smad2 phosphorylation, which coincided with restoration of the proliferation rate in Smad2-overexpressing cells. ELISA of TGF-ß release did not show any differences between genotypes. The cell cycle inhibitors, p15 and p21, showed significant upregulation in Smad2-overexpressing cells compared to wild-type controls. Moreover, junctional epithelium of the transgenic mice showed increased expression of P-Smad2, p15 and p21. CONCLUSION: The signaling activation triggered by overexpression of Smad2 was dependent on TGF-ß type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.

Assessment of the plasma/serum IgG test to screen for periodontitis.

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients.

Smad2 decelerates re-epithelialization during gingival wound healing.

During periodontal regeneration, inhibition of gingival downgrowth is necessary to promote migration of mesenchymal cells into the defects. Transforming growth factor (TGF)-ß is a pleiotropic cytokine that has numerous cell functions, including regulation of epithelial growth. Recent studies have shown that Smad2, a downstream transcription factor of TGF-ß, plays crucial roles in wound healing in the epithelia. Therefore, we investigated the effects of Smad2 overexpression on re-epithelialization of gingival wounds. Transgenic mice overexpressing smad2 driven by the keratin 14 promoter (k14-smad2) were confirmed to have significant Smad2 phosphorylation in gingival basal epithelia. Punch wounds were made in the palatal gingiva, and wound healing was assessed histologically for 7 days. Re-epithelialization was significantly retarded on day 2, while collagen deposition was enhanced on day 7 in k14-smad2 compared with wild-type mice. Moreover, expression of keratin 16 (K16), an indicator of keratinocyte migration, was significantly inhibited in wound-edge keratinocytes in k14-smad2. The inhibition of K16 coincided with the induction of Smad2 in the corresponding epithelia, while BrdU incorporation was unaffected. These results indicated that Smad2 has inhibitory effects in regulating keratinocyte migration during gingival wound healing. TGF-ß/Smad2 signaling mediating alteration of K16 expression must be tightly regulated during periodontal regeneration.

Immune responses to porphyromonas gingivalis infection suppress systemic inflammatory response in experimental murine model.

Periodontitis is a localized infectious disease caused by periodontopathic bacteria such as Porphyromonas gingivalis (P. gingivalis), and the severity correlates to significance of immune responses. Recently, it has been reported that periodontitis is associated with the development of systemic disease such as diabetes and atherosclerosis because of increasing invasion of oral pathogens to the circulation. However, the association between local and systemic infectious responses is still unclear. In the present study, we examined the differences of biological responses in animals with or without bacterial infection. After Balb/c mice were infected subcutaneously with live P. gingivalis W83, serum, skin and liver were collected according to experimental protocol. The skin and liver tissues were observed pathologically by haematoxylin-eosin staining, and serum IL-6 levels were measured using ELISA method. Throughout the experimental period, conditions of the mice were observed continuously. As expected, severe infiltration of leukocytes were observed at inflamed skin corresponding to the number of bacterial challenges. Although no inflammatory appearance of skin was observed, serum IL-6 levels were increased dramatically (P <0.01, Student's t-test) and liver tissues were injured in the mice without bacterial challenge. Interestingly, although severe inflammatory appearance of the skin was observed, serum IL-6 levels were not increased and no inflammatory responses were observed in the liver of the 3-times bacterially challenged group. Importantly, immunoglobulin G against P. gingivalis W83 was detected in the blood of mice with 3-times bacterial challenge corresponding to improvement of weight loss and survival. In conclusion, although multiple infections develop severe localized inflammation, the immune system should be sufficient to protect the systemic inflammatory responses.

Discovery of a patient with strongly suspected bullous pemphigoid in a ward by oral health care providers.

OBJECTIVES: Oral health care providers may discover systemic diseases incidentally from signs observed in the oral cavity. Here, we report a case in which oral health care providers in a hospital discovered a patient with strongly suspected bullous pemphigoid (BP), which is a relatively rare but important disease, in a ward. METHODS: The patient was a 78-year-old Japanese woman admitted to our hospital because of severe Alzheimer's disease. We discovered recurrent ulcers in the oral mucosa and skin when performing oral care in her ward. Biopsy could not be performed safely because of involuntary biting. We performed blood tests for anti-BP180-NC16a antibody, which is autoantibody specific for BP. RESULTS: The patient had a very high anti-BP180-NC16a antibody titre. We consulted a dermatologist regarding her clinical course and the clinical features of the oral mucosa and skin along with blood test results. BP was very strongly suspected. DISCUSSION: In cases in which oral health care providers suspect their patients may have BP, appropriate examination and provision of information to the doctor are important. Oral health care providers should have knowledge about systemic diseases, the signs of which appear in oral cavity to avoid missing important systemic diseases.

FGF-2 stimulates periodontal regeneration: results of a multi-center randomized clinical trial.

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

Antigenic group II chaperonin in Methanobrevibacter oralis may cross-react with human chaperonin CCT.

Methanobrevibacter oralis is an archaeal species frequently isolated from sites of severe periodontitis. However, its pathogenic roles remain unclear. Here, we aimed to isolate group II chaperonin from M. oralis and examine its antigenicity. The genes encoding two chaperonin subunits (Cpn-1 and Cpn-2) were cloned from M. oralis using polymerase chain reaction and genome walking procedures. Recombinant proteins Cpn-1 and Cpn-2 were generated, and the reactivities of sera from patients with periodontitis were examined by Western immunoblotting. The open reading frames of Cpn-1 and Cpn-2 genes consisted of 1641 and 1614 base pairs, respectively. Putative ATP-binding domains conserved among the chaperonin family were observed in both genes. The deduced amino acid sequences of the two genes showed 28.8-40.0% identity to each of the subunits of human CCT (CCT1-8). Thirty and 29 of 36 patients' sera reacted with the recombinant Cpn-1 and recombinant Cpn-2, respectively. Western immunoblotting using antiserum against human CCT subunits indicated that anti-CCT3 and anti-CCT8 antibodies recognized recombinant Cpn-1. In addition, anti-CCT1, CCT3, CCT6, and CCT8 antibodies recognized an antigen of approximately 60 kDa in M. oralis. The results suggested that the chaperonin subunits of M. oralis were antigenic molecules that were recognized by periodontitis patients and that may cross-react with human chaperonin CCT.

Genetic risk factors for periodontitis in a Japanese population.

Genetic variants at multiple loci have been shown to be associated with susceptibility to periodontitis. To better assess the genetic risk factors for periodontitis, we performed a case-control study in 319 Japanese individuals with periodontitis (172 aggressive and 147 chronic disease) and 303 race-matched healthy control individuals. Thirty-five functional gene polymorphisms that had been previously associated with immune responses were genotyped. For all gene polymorphisms tested, no significant differences were observed in the allele frequencies of persons with aggressive, chronic, and combined (aggressive and chronic) periodontitis, compared with control individuals. Multiple logistic regression analysis revealed a significant association of the vitamin D receptor +1056 T/C polymorphism with susceptibility to chronic periodontitis, after adjustment for age, gender, and smoking status (P = 0.002). These results suggest that none of the polymorphisms tested was strongly associated with periodontitis in a Japanese population. However, the vitamin D receptor +1056 polymorphism may be related to chronic periodontitis.

Human leukocyte histocompatibility antigen class II-induced cytokines from human gingival fibroblasts promote proliferation of human umbilical vein endothelial cells: potential association with enhanced angiogenesis in chronic periodontal inflammation.

BACKGROUND AND OBJECTIVE: The role of human leukocyte histocompatibility antigen (HLA) class II molecules on non-antigen-presenting cells has been a matter of controversy. We previously reported that HLA-II molecules on human gingival fibroblasts (GF) do not present antigens, but transduce signals into the cells, resulting in the expression of several cytokines, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T-cell expressed and secreted (RANTES) and IL-8. However, the exact role of these cytokines, as well as other cytokines which are potentially secreted from GF, in the pathogenesis of chronic periodontal inflammation is not fully understood. The aim of this study was to observe the effects of HLA-II-induced cytokines on the proliferation of human umbilical vein endothelial cells (HUVEC). MATERIAL AND METHODS: Antibody-based cytokine-microarray analyses were performed to detect potential cytokines associated with angiogenesis. Next, cytokine productivity was confirmed by quantitative methods. Then, cell proliferation assay was performed to see whether these cytokines promoted the proliferation of HUVEC. RESULTS: Besides IL-6, MCP-1, RANTES and IL-8, growth-related gene product (GRO) was newly identified as an HLA-II-induced cytokine released from GF. This was confirmed by a quantitative method. Cell culture supernatant from HLA-II-stimulated GF cultures promoted the growth of HUVEC. Addition of anti-IL-8 neutralizing antibody, anti-CXC receptor (CXCR)1 antibody and anti-MCP-1 antibody inhibited the growth of HUVEC in a dose-dependent manner, while addition of anti-GROalpha antibody did not. CONCLUSION: The HLA-II-induced IL-8, via CXCR1, as well as MCP-1 from GF, promotes endothelial cell proliferation, which is possibly associated with enhanced angiogenesis in chronic periodontal lesions.

Porphyromonas gingivalis fimbriae-dependent interleukin-6 autocrine regulation by increase of gp130 in endothelial cells.

BACKGROUND AND OBJECTIVE: Local persistent infection by Porphyromonas gingivalis leads to inflammatory systemic diseases, such as atherosclerosis. We have reported previously that avirulent P. gingivalis fimbriae-dependent invasion into endothelial cells might be involved in progression of atherosclerosis. Although interleukin-6 (IL-6) regulates progression of atherosclerosis, little is known about the relationship of P. gingivalis fimbriae-dependent invasion to IL-6 regulation in endothelial cells. MATERIAL AND METHODS: We examined the secretion of IL-6 and the expression of the IL-6 signal transducer gp130 in human umbilical vein endothelial cells (HUVEC) infected with the wild-type FDC381 strain of P. gingivalisand a fimbriae-deficient mutant (fimA) by enzyme-linked immunosorbent assay, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry (fluorescence-activated cell sorting, FACS) analysis. RESULTS: Coculture of HUVEC with P. gingivalis resulted in increase of IL-6 secretion at 24 h postinfection. Interestingly, the increase was inhibited significantly in HUVEC infected with the P. gingivalis fimA mutant. In addition, the increase of IL-6 secretion induced by P. gingivalis infection was significantly impaired by the meiosis specific kinase 1 inhibitor, PD98059, or the nuclear factor kappaB inhibitor, Bay11-7082. Furthermore, we demonstrated that gp130 expression increased with P. gingivalis infection. Importantly, gp130 expression was significantly impaired by P gingivalis fimA mutant infection compared with wild-type P. gingivalis infection, as assessed by both quantitative RT-PCR and FACS analysis. CONCLUSION: Our findings indicate that P. gingivalis fimbriae are important factors in the autocrine regulation of IL-6, by increasing gp130 in endothelial cells.

Focal adhesion kinase mediates human leukocyte histocompatibility antigen class II-induced signaling in gingival fibroblasts.

BACKGROUND AND OBJECTIVE: The role of human leukocyte antigen class II molecules on nonantigen-presenting cells has been a matter of controversy. We previously reported that human leukocyte antigen class II molecules on human gingival fibroblasts do not present antigens, but transduce signals into the cells by making a complex with antigenic peptide T-cell receptor or by stimulating cell surface human leukocyte antigen-DR molecules with human leukocyte antigen-DR antibody (L243), which mimics the formation of the human leukocyte antigen class II-antigenic peptide T-cell receptor complex, resulting in the expression of several cytokines. The aim of this study was to detect human leukocyte antigen class II-associated molecules mediating human leukocyte antigen class II-induced signals into the cells. MATERIAL AND METHODS: Antibody-based protein-microarray analysis was performed to detect activated signaling molecules in gingival fibroblasts stimulated via human leukocyte antigen class II molecules. Then, we examined if these molecules structurally associate with human leukocyte antigen class II and actually transduce signals into the cells. RESULTS: Stimulation of human leukocyte antigen class II on gingival fibroblasts by L243 resulted in enhanced phosphorylation of focal adhesion kinase. Focal adhesion kinase was co-immunoprecipitated with human leukocyte antigen-DR by L243. Stimulation of gingival fibroblasts with L243 induced phosphorylation of focal adhesion kinase. Luteolin, a putative focal adhesion kinase inhibitor, suppressed phosphorylation of focal adhesion kinase and dose dependently inhibited human leukocyte antigen class II-induced cytokine production. CONCLUSION: Focal adhesion kinase is structurally associated with human leukocyte antigen-DR and mediates human leukocyte antigen class II-induced signals in gingival fibroblasts.

Relationship of periodontal infection to serum antibody levels to periodontopathic bacteria and inflammatory markers in periodontitis patients with coronary heart disease.

Several reports have demonstrated a possible association of periodontal infections with coronary heart disease (CHD) by elevated antibody titre to periodontopathic bacteria in CHD patients compared with non-diseased controls. Although each periodontopathic bacterium may vary in virulence for periodontitis and atherosclerosis, antibody response to multiple bacteria in CHD patients has not been understood fully. Therefore, serum levels of antibody to 12 periodontopathic bacteria together with other atherosclerotic risk markers were compared among 51 patients with CHD, 55 patients with moderate to severe chronic periodontitis and 37 healthy individuals. The antibody response was the most prevalent for Porphyromonas gingivalis, a major causative organism, in CHD as well as periodontitis patients. However, antibody positivity was different between CHD and periodontitis if the response was analysed for two different strains of P. gingivalis, namely FDC381 and Su63. While periodontitis patients were positive for both P. gingivalis FDC381 and Su63, a high frequency of antibody positivity for P. gingivalis Su63 but not for FDC381 was observed in CHD patients. The results indicate that the presence of particular periodontopathic bacteria with high virulence may affect atherogenesis. Identifying the virulence factors of P. gingivalis Su63 may gain insight into the new therapeutic modality for infection-induced deterioration of atherosclerosis.

Thiazolidinedione (pioglitazone) blocks P. gingivalis- and F. nucleatum, but not E. coli, lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) production in adipocytes.

An elevated level of C-reactive protein (CRP) predicts the future development of coronary heart disease. Periodontitis appears to up-regulate CRP. CRP is produced by hepatocytes in response to interleukin-6 (IL-6). A major source of IL-6 in obese subjects is adipocytes. We hypothesized that lipopolysaccharide (LPS) from periodontal pathogens stimulated adipocytes to produce IL-6, and that the production was suppressed by the drugs targeted against insulin resistance, thiazolidinedione (pioglitazone), since this agent potentially showed an anti-inflammatory effect. Mouse 3T3-L1 adipocytes were stimulated with E. coli, P. gingivalis, and F. nucleatum LPS. The IL-6 concentration in culture supernatants was measured. All LPS stimulated adipocytes to produce IL-6. Although pioglitazone changed adipocyte appearance from large to small, and completely suppressed P. gingivalis and F. nucleatum LPS-induced IL-6 production, E. coli LPS-induced IL-6 production was not efficiently blocked. Thus, pioglitazone completely blocked periodontal-bacteria-derived LPS-induced IL-6 production in adipocytes, a major inducer of CRP.

Impairment of gingival fibroblast adherence by IL-6/sIL-6R.

Interleukin-6 (IL-6) binds to human gingival fibroblasts (HGF) in the presence of a soluble form of IL-6 receptor (sIL-6R). We investigated the effects of IL-6 on the functions of HGF in the presence of sIL-6R. HGF changed their morphology from spindle-shaped to round, and detached from the culture dish by stimulation with IL-6/sIL-6R. In this condition, a signal transducer gp130 and a transcription factor Stat3 were phosphorylated, resulting in activation of transcription factors Stat3 and C/EBPbeta. Cytoskeletal beta-actin and adhesion molecule integrin-alpha5, a subunit of alpha5beta1 integrin (VLA-5), were found to possess potential binding domains for these transcription factors in their promoters. Accumulation of beta-actin and integrin-alpha5 mRNA decreased, contrary to the expectation of the induction of gene transcription. Furthermore, the decrease in their mRNAs was associated with reduced expression of both actin and VLA-5 proteins. These results suggest that the expression of VLA-5 and actin was down-regulated in HGF through an IL-6 signaling pathway, resulting in impairment of HGF adherence.

Heterogeneity of host immunological risk factors in patients with aggressive periodontitis.

BACKGROUND: The pathogenesis of early-onset periodontitis (EOP) can be explained by various host risk factors. Previous studies have focused on a single (among many possible) immunological risk factor and the association among the factors has not been assessed. We comprehensively investigated the associations among multiple host immunological risk factors in EOP patients to further elucidate their role in the pathogenesis of EOP. METHODS: Sixty-eight EOP patients (50 generalized EOP, 18 localized EOP), 51 EOP-suspected patients (S-EOP), 43 adult periodontitis (AP) patients, and 36 periodontally healthy subjects (HS) participated in this cross-sectional study. We examined peripheral neutrophil functions, phenotypic and functional characterization of peripheral lymphocytes (lymphocyte subsets, T-cell proliferative activity), cytokine productivity (interleukin [IL]-1, IL-2, tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma, IL-4 and IL-6), serum immunoglobulin G (IgG) antibody titers against 12 periodontal bacteria, and HLA class II genotypes. RESULTS: G-EOP, S-EOP, and AP patient groups showed significantly lower percentages of pan T cells and CD8-positive cells (P < 0.02) compared with the HS group. L-EOP patients showed depressed IL-4 and TNF-alpha productivity compared with the HS group (P < 0.02). The EOP group showed significantly elevated antibody levels against Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum compared with the HS group (P < 0.05). The frequency with DQB1*0503 was significantly higher in the EOP patient group than the HS group (P = 0.045) due to the higher frequency in L-EOP patients than the HS group (P = 0.035). There were wide interindividual variations in each of the tests among patient and HS groups; however, EOP patients showed wider intradiagnostic group variations in certain host defensive cell functions than the other groups. There were some EOP patients who showed extremely low or high values in some tests; the EOP patients could be further divided into subgroups according to their host defensive and immunological profiles. However, there was heterogeneity in some of the other host immunological tests even in the subgroups. CONCLUSIONS: The association of host immunological risk factors in EOP patients is widely varied and more complex than previously thought. These results indicate the difficulty of explaining the pathogenesis of EOP based on a single host risk factor and also emphasize the importance of critical assessment of not only EOP patient groups, but also individual patients.

Phenytoin and cyclosporin A suppress the expression of MMP-1, TIMP-1, and cathepsin L, but not cathepsin B in cultured gingival fibroblasts.

BACKGROUND: Fibroblasts are known not only to synthesize and secrete extracellular matrix proteins, but also to degrade them for connective tissue remodeling. Drug-induced gingival overgrowth is characterized by a massive accumulation of extracellular matrix components in gingival connective tissues. Although some previous reports suggested that causative drugs stimulated the fibroblast proliferation, the results are not conclusive yet. In this study, we hypothesized that drug-induced gingival overgrowth could be a consequence of impaired ability of matrix degradation rather than an enhanced proliferation of gingival fibroblasts induced by these drugs. METHODS: Normal human gingival fibroblasts were cultured with or without either 20 microg/ml of phenytoin or 200 ng/ml of cyclosporin A. Total RNA and cellular proteins were collected every day for RT-PCR analyses and for measuring lysosomal enzyme activity. In addition, an immunohistochemical study was performed to detect lysosomal enzymes in cells from enlarged gingiva of the patients with phenytoin-induced gingival overgrowth. RESULTS: RT-PCR analyses revealed that these drugs suppressed the expression of MMP-1, TIMP-1, and cathepsin L, but not that of cathepsin B in a time-dependent manner. Then, we measured the activity of lysosomal enzymes and cathepsin B and L. The results indicated that although cathepsin B activity was not observed to be impaired, regardless of the drugs used in these cells, both total and active forms of combined activity of cathepsins B and L were suppressed in a time-dependent manner. CONCLUSIONS: The results indicate that, besides suggested effects of these drugs on gingival fibroblasts and/or on accumulated cells in the gingival tissues, extracellular matrix-degrading ability, particularly that by cathepsin L, is also suppressed by cyclosporin A and phenytoin in gingival fibroblasts, and that lysosomal enzyme plays an important role in the pathogenesis of drug-induced gingival hyperplasia.

Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis.

Porphyromonas gingivalis, a putative pathogen in human periodontal disease, possesses a 60-kDa heat shock protein (hsp60, GroEL). The GroEL homologs are known to be key molecules in auto-immune reactions because of the sequence similarity with human hsp60. In this study, B-cell epitopes on P. gingivalis GroEL (PgGroEL) were analyzed by both Western immunoblotting with truncated PgGroEL and by the multi-pin synthetic peptide approach. To examine auto-antibody production in periodontitis patients, Western immunoblotting with human gingival fibroblasts was performed. Deletion mutants were constructed from the cloned PgGroEL gene (P. gingivalis groEL), and four C-terminal truncated PgGroEL and one N-terminal truncated PgGroEL were prepared from the deletants. Sera from periodontitis patients reacted with all truncated PgGroEL used in this study. The results suggest that the B-cell epitopes were overlaid throughout PgGroEL. To determine the detailed locations of the B-cell epitope, 84 decapeptides covering the entire PgGroEL were synthesized and the serum IgG response to the peptides was examined. Epitope mapping using the synthetic peptides confirmed that the B-cell epitopes were overlaid throughout the length of PgGroEL and revealed that highly conserved peptides between PgGroEL and human hsp60 were recognized by the serum antibodies. Immuno-reactivity against human gingival fibroblasts was examined with sera from 30 periodontitis patients and 10 periodontally healthy subjects. IgG antibody against the 65-kDa antigen in human gingival fibroblasts (same molecular mass as human hsp60) was detected in two patients. Although IgG production against human hsp60 may be rare case in periodontitis patients, the results of epitope mapping demonstrated the potential of PgGroEL to cause the cross-reactions with human hsp60.

Molecular characterization of the hlyX-like gene of Actinobacillus actinomycetemcomitans Y4.

We isolated and characterized a possible regulatory gene, designated actX gene, from Actinobacillus actinomyctemcomitans Y4, which defined the Actinobacillus pleuropneumoniae hlyX-like regulatory gene. DNA sequence analysis for plasmid clone pKM317 containing a 1.6-kb DNA insert indicated an open reading frame encoding a polypeptide of 257 amino acid residues. Analysis of the deduced amino acid sequence showed the presence of five characteristic cysteine residues in the N-terminal region and a putative DNA binding residue in the C-terminal region, indicating that actX might belong to a regulatory gene family. Escherichia coli DH5alpha and a mutant strain JRG1728 transformed by plasmid carrying actX manifested apparent hemolytic activity on sheep blood agar and grew anaerobically, although the original strains did not.