RESUMO
BACKGROUND AND OBJECTIVES: Previous reports suggest that several serum biomarkers play roles in the pathogenesis, inflammatory response, and oxidative stress in periodontitis caused by bacterial infections, linking chronic periodontitis to atherosclerotic vascular disease (ASVD). The aim of this preliminary study was to investigate, in a Japanese cross-sectional community survey, potential serum biomarkers of periodontitis that are associated with ASVD and chronic periodontitis. MATERIAL AND METHODS: The study cohort included a total of 108 male subjects who underwent annual health examinations. Serum biomarkers (high-sensitivity C-reactive protein [hs-CRP], proprotein convertase subtilisin/kexin type 9 [PCSK9], interleukin-6, tumor necrosis factor-α, soluble CD14, myeloperoxidase, matrix metalloproteinase-3, adiponectin, total bilirubin [TBIL], and serum lipids) were analyzed to determine their association (if any) with periodontal parameters. Aortic stiffness was evaluated using the brachial-ankle aortic pulse wave velocity (PWV) index and the cardio-ankle vascular index (CAVI). RESULTS: The concentrations of PCSK9 and hs-CRP were increased (P = .001 and .042, respectively), and the concentration of TBIL was decreased (P = .046), in subjects with periodontal disease (determined as a probing depth of ≥4 mm in at least one site) compared with periodontally healthy subjects. The ratio of low-density lipoprotein cholesterol (LDL-C) to high-density lipoprotein cholesterol and the concentrations of triglycerides, remnant-like particles-cholesterol, and oxidized LDL were elevated in subjects with periodontal disease compared with periodontally healthy subjects (P = .038, .007, .002, and .049, respectively). Multivariate regression analyses indicated that the number of sites with a pocket depth of ≥4 mm was associated with the concentration of PCSK9 and inversely associated with the concentration of TBIL independently (standardized ß = .243, P = .040; standardized ß = -.443, P = .0002; respectively). Analysis of receiver operating characteristic curves of PCSK9 indicated moderate accuracy for predicting the presence of disease sites (probing depth ≥ 4 mm) (area under the curve = 0.740). No significance in the values of PWV and CAVI was observed between subjects with periodontal disease and periodontally healthy subjects. CONCLUSION: In Japanese male subjects, the concentrations of serum PCSK9 and TBIL were correlated with periodontal parameters. Moreover, PCSK9 could be a candidate biomarker for diagnosing chronic periodontitis, and may also have potential to evaluate the risk for periodontitis to cause ASVD. Longitudinal studies of larger populations are necessary to confirm the exact association of periodontitis with increased serum PCSK9 and decreased TBIL.
Assuntos
Bilirrubina/sangue , Periodontite Crônica/sangue , Pró-Proteína Convertase 9/sangue , Adiponectina/sangue , Adulto , Povo Asiático , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Periodontite Crônica/diagnóstico , Periodontite Crônica/enzimologia , Estudos de Coortes , Estudos Transversais , Humanos , Interleucina-6/sangue , Japão , Receptores de Lipopolissacarídeos/sangue , Lipoproteínas/sangue , Estudos Longitudinais , Masculino , Metaloproteinase 3 da Matriz/sangue , Pessoa de Meia-Idade , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND AND OBJECTIVE: In periodontitis, chronic infection by periodontopathic bacteria induces uncontrolled inflammation, which leads to periodontal tissue destruction. Human gingival epithelial cells (HGECs) constitute a critical first line of defense against periodontopathic bacteria, both as a physical barrier and as regulators of inflammation. Resveratrol, a polyphenol found in grapes and red wine, reportedly has anti-inflammatory properties. Therefore, we investigated the effects of resveratrol on the Porphyromonas gingivalis-induced inflammatory responses of HGECs and their mechanism. MATERIAL AND METHODS: We stimulated the HGEC line, epi 4, with live or heat-killed P. gingivalis in the presence of resveratrol, and analyzed expressions of the interleukin-8, monocyte chemoattractant protein-1 and interleukin-1ß genes. We determined the involvement of SIRT1 in the effect of resveratrol using sirtinol (a SIRT1 inhibitor) or SIRT1 knockdown. We also examined whether the effects were mediated by activation of AMP-activated kinase, suppression of reactive oxygen species, or inhibition of nuclear factor-κB (NF-κB). RESULTS: Resveratrol treatment decreased the expression of inflammatory cytokines and slightly increased the expression of SIRT1. However, neither SIRT1 inhibition nor SIRT1 knockdown counteracted its anti-inflammatory effects. Although resveratrol did not affect AMP-activated kinase activation or reactive oxygen species production, it slightly suppressed NF-κB translocation when cells were stimulated with heat-killed P. gingivalis. CONCLUSION: Resveratrol suppressed the inflammatory responses of P. gingivalis-stimulated HGECs, probably by inhibiting NF-κB signaling but independent of SIRT1.
Assuntos
Gengiva , Quimiocina CCL2 , Células Epiteliais , Humanos , Interleucina-8 , NF-kappa BRESUMO
BACKGROUND AND OBJECTIVE: Elderly people exhibit increased susceptibility to a number of autoimmune and infectious diseases, such as periodontitis. Although aging is reportedly associated with a decline in immune function, age-related alterations in periodontal tissue have remained elusive. In the present study, we comprehensively analyzed the effect of aging on the expression of selected genes using mouse gingival fibroblasts. MATERIAL AND METHODS: Gingival fibroblasts derived from young (8 wk of age) and old (≥ 24 mo of age) C57BL/6 mice were stimulated with Porphyromonas gingivalis lipopolysaccharide or live P. gingivalis strain W83. Expression of cytokines/chemokines, innate immune receptors, growth factors, matrix metalloproteinases, tissue inhibitors of metalloproteinases and osteoclastogenesis-related molecules were evaluated using real-time polymerase chain reaction and ELISA for interleukin-6 and transforming growth factor-ß1. RESULTS: Gingival fibroblasts derived from old mice exhibited decreased gene expression of Il-6, Cxcl1, Tlr2, Tlr4, Irak3 (IRAK-M), Kgf, Timp1, Timp3 and Rankl under resting conditions, whereas the expression levels of Tgfß1, Mmp3, Mmp13 and Opg were increased. Age-related differences were also detected at the protein level. Although P. gingivalis W83 upregulated Vegf, Fgf-2 and Bmp2 expression in both young and old gingival fibroblasts, the stimulatory effect on these genes was significantly lower in old gingival fibroblasts. CONCLUSION: Our findings demonstrated that aging altered the expression of a number of genes in gingival fibroblasts. Thus, alterations in the balance of these molecules could play a critical role in periodontitis progression in the elderly.
Assuntos
Envelhecimento/genética , Fibroblastos/microbiologia , Gengiva/microbiologia , Porphyromonas gingivalis/imunologia , Envelhecimento/imunologia , Animais , Proteína Morfogenética Óssea 2/análise , Quimiocina CXCL1/análise , Fator 2 de Crescimento de Fibroblastos/análise , Fator 7 de Crescimento de Fibroblastos/análise , Fibroblastos/imunologia , Gengiva/imunologia , Imunidade Inata/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/análise , Quinases Associadas a Receptores de Interleucina-1/análise , Interleucina-6/análise , Lipopolissacarídeos/imunologia , Masculino , Metaloproteinase 13 da Matriz/análise , Metaloproteinase 3 da Matriz/análise , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/fisiologia , Osteoprotegerina/análise , Ligante RANK/análise , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-3/análise , Receptor 4 Toll-Like/análise , Fator de Crescimento Transformador beta1/análise , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
BACKGROUND AND OBJECTIVE: T and B cells are known to be involved in the disease process of periodontitis. However, the role of natural killer T cells in the pathogenesis of periodontitis has not been clarified. MATERIALS AND METHODS: To examine the role of these cells, C57BL/6J (wild-type), CD1d(-/-) and α-galactosylceramide (αGC)-stimulated wild-type mice were orally infected with Porphyromonas gingivalis strain W83. RESULTS: Apart from CD1d(-/-) mice, the level of alveolar bone resorption was elevated by the infection and was further accelerated in αGC-stimulated mice. The infection induced elevated levels of serum amyloid A and P. gingivalis-specific IgG in the sera, although the degree of elevation was much smaller in the CD1d(-/-) mice. Infection-induced RANKL elevation was only observed in αGC-stimulated mice. Although the cytokines produced by splenocytes were mainly T-helper 1 type in wild-type mice, those in αGC-stimulated mice were predominantly T-helper 2 type. In the liver, the infection demonstrated no effect on the gene expression for interferon-γ, interleukin-4 and RANKL except αGC-stimulated mice in which the infection upregulated the gene expressions. CONCLUSION: This study is the first to show that natural killer T cells upregulated systemic and local inflammatory responses induced by oral infection with P. gingivalis, thereby contributing to the progression of alveolar bone resorption.
Assuntos
Perda do Osso Alveolar/imunologia , Infecções por Bacteroidaceae/imunologia , Células Matadoras Naturais/imunologia , Periodontite/microbiologia , Porphyromonas gingivalis/imunologia , Perda do Osso Alveolar/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos CD1d/imunologia , Galactosilceramidas/farmacologia , Imunoglobulina G/sangue , Inflamação/imunologia , Interferon gama/análise , Interleucina-4/análise , Células Matadoras Naturais/microbiologia , Fígado/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Periodontite/imunologia , Ligante RANK/análise , Ligante RANK/efeitos dos fármacos , Proteína Amiloide A Sérica/análise , Baço/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
BACKGROUND AND OBJECTIVE: Periodontal infection affects atherosclerotic diseases, such as coronary heart diseases. Mouse models have revealed that oral infection with Porphyromonas gingivalis induces changes in inflammatory- and lipid metabolism-related gene expression, regardless of the development of atherosclerotic lesions. However, the serum protein expression profile in the oral infection model has not been investigated. The present study aimed to analyse the effect of oral infection with P. gingivalis on the expression levels of multiple cytokines in the serum in apolipoprotein E-deficient mice by using a cytokine antibody array. MATERIAL AND METHODS: C57BL/6.KOR-Apoe(shl) mice were orally infected with P. gingivalis five times at 3 day intervals and were then killed. Splenocytes were isolated and analysed for proliferative activity and immunoglobulin G (IgG) production in response to in vitro restimulation with P. gingivalis. The expression levels of various cytokines in the sera were analysed using a mouse antibody array glass chip. RESULTS: Splenocytes from P. gingivalis-infected mice demonstrated significantly greater proliferation and IgG production in response to P. gingivalis compared with those from sham-infected mice. Antibody array analysis revealed the selective upregulation of matrix metalloproteinase 3, intercellular adhesion molecule 1, insulin-like growth factor binding protein 2 and chemokine (C-X-C motif) ligand 7 and the downregulation of interleukin-17, tumor necrosis factor-α and L-selectin. CONCLUSION: These data demonstrate that oral infection with P. gingivalis induces alterations in systemic cytokine production. These cytokines could play roles in the development not only of periodontitis but also of atherosclerosis.
Assuntos
Infecções por Bacteroidaceae/imunologia , Citocinas/sangue , Doenças da Boca/microbiologia , Porphyromonas gingivalis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Apolipoproteínas E/genética , Proliferação de Células , Quimiocinas CXC/sangue , Modelos Animais de Doenças , Imunoglobulina G/sangue , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Molécula 1 de Adesão Intercelular/sangue , Interleucina-17/sangue , Interleucina-6/sangue , Selectina L/sangue , Masculino , Metaloproteinase 3 da Matriz/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Doenças da Boca/imunologia , Baço/citologia , Baço/imunologia , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND AND OBJECTIVE: Recent studies have revealed that negative regulatory molecules, including interleukin-1 receptor-associated kinase-M (IRAK-M), control the overactivation of Toll-like receptor (TLR) signaling. The role of IRAK-M in human gingival epithelial cells (HGECs), which express TLRs, remains unclear. The present study examined the role of IRAK-M on interleukin-8 and macrophage chemoattractant protein-1 (MCP-1) expression in HGECs stimulated with Porphyromonas gingivalis and TLR ligands. MATERIAL AND METHODS: Primary HGECs and an SV40 T-antigen-immortalized HGEC line (epi 4) were stimulated with live or heat-killed P. gingivalis, P. gingivalis lipopolysaccharide or the synthetic lipopeptide PAM(3)CSK(4), and subsequent expression of IRAK-M, interleukin-8 and MCP-1 was evaluated at the mRNA and protein levels. The effects of IRAK-M on interleukin-8 and MCP-1 expressions were evaluated by IRAK-M-specific RNA interference (RNAi)-based loss-of-function assay. RESULTS: All tested stimulants up-regulated the expression of IRAK-M in HGECs. The P. gingivalis lipopolysaccharide or PAM(3)CSK(4) increased MCP-1 expression, whereas live P. gingivalis down-regulated the MCP-1 expression in HGECs. However, IRAK-M RNAi increased the expression of MCP-1 irrespective of up- or down-regulation mediated by the respective stimulants. Interleukin-8 gene expression, up-regulated by all tested stimulants, was further enhanced by IRAK-M RNAi. In contrast, IRAK-M RNAi had no effect on the interleukin-8 protein levels, irrespective of the stimulant, indicating that post-translational modification, not IRAK-M, controls interleukin-8 protein expression. CONCLUSION: Interleukin-1 receptor-associated kinase-M appeared to have distinct regulatory roles on the interleukin-8 and MCP-1 produced by HGECs, further suggesting an important role for interleukin-8 in the immune response to periodontopathic bacteria.
Assuntos
Quimiocina CCL2/imunologia , Gengiva/imunologia , Quinases Associadas a Receptores de Interleucina-1/imunologia , Interleucina-8/imunologia , Porphyromonas gingivalis/imunologia , Linhagem Celular , Células Cultivadas , Regulação para Baixo/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Inativação Gênica , Gengiva/citologia , Gengiva/microbiologia , Humanos , Inositol Polifosfato 5-Fosfatases , Quinases Associadas a Receptores de Interleucina-1/genética , Interleucina-8/metabolismo , Ligantes , Lipopeptídeos/imunologia , Lipopolissacarídeos/imunologia , Monoéster Fosfórico Hidrolases/análise , Processamento de Proteína Pós-Traducional/imunologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/análise , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Regulação para Cima/imunologiaRESUMO
BACKGROUND AND OBJECTIVE: Although an elevation in the concentration of high-sensitivity C-reactive protein (hs-CRP) as a result of periodontal infection may account for an increased risk of developing coronary heart disease (CHD), the effect of periodontal infection on the level of hs-CRP in an otherwise healthy Japanese population has not yet been reported. The aim of the present study was to confirm, on a larger scale, our previous pilot study findings that both chronic periodontitis and subsequent periodontal treatment alter the serum levels of C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). MATERIAL AND METHODS: The concentrations of serum hs-CRP, IL-6 and TNF-alpha were measured in 78 periodontitis patients at baseline and at re-assessment, and in 40 periodontally healthy subjects at the time of examination. RESULTS: The concentrations of hs-CRP and IL-6 in the sera of periodontitis patients were significantly higher than those in control subjects. By contrast, the concentration of TNF-alpha was significantly lower in periodontitis patients than in control subjects. Whereas periodontal treatment decreased the levels of serum hs-CRP and IL-6, no such effect was observed for TNF-alpha. When the patients were subdivided into four groups according to their initial concentration of hs-CRP, only the CRP and IL-6 concentrations of the highest quartile group showed a significant reduction following periodontal treatment. No significant difference in the initial clinical parameters was observed in any quartile. CONCLUSION: Although periodontal infection does affect the concentration of hs-CRP and IL-6 in serum, a subgroup of patients exist who are highly susceptible to an increased risk of CHD associated with periodontitis, suggesting that there may be subjects who have an elevated risk of CHD independent of susceptibility to periodontal tissue destruction per se.
Assuntos
Periodontite Crônica/sangue , Doença das Coronárias/etiologia , Mediadores da Inflamação/sangue , Regulação para Cima/fisiologia , Perda do Osso Alveolar/sangue , Perda do Osso Alveolar/classificação , Proteína C-Reativa/análise , Periodontite Crônica/classificação , Periodontite Crônica/terapia , Raspagem Dentária , Suscetibilidade a Doenças , Feminino , Seguimentos , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Perda da Inserção Periodontal/sangue , Perda da Inserção Periodontal/classificação , Bolsa Periodontal/sangue , Bolsa Periodontal/classificação , Fatores de Risco , Aplainamento Radicular , Fumar , Retalhos Cirúrgicos , Fator de Necrose Tumoral alfa/análiseRESUMO
In humans, ten Toll-like receptor (TLR) paralogues sense molecular components of microbes, initiating the production of cytokine mediators that create the inflammatory response. Using N-ethyl-N-nitrosourea, we induced a germline mutation called Lps2, which abolishes cytokine responses to double-stranded RNA and severely impairs responses to the endotoxin lipopolysaccharide (LPS), indicating that TLR3 and TLR4 might share a specific, proximal transducer. Here we identify the Lps2 mutation: a distal frameshift error in a Toll/interleukin-1 receptor/resistance (TIR) adaptor protein known as Trif or Ticam-1. Trif(Lps2) homozygotes are markedly resistant to the toxic effects of LPS, and are hypersusceptible to mouse cytomegalovirus, failing to produce type I interferons when infected. Compound homozygosity for mutations at Trif and MyD88 (a cytoplasmic TIR-domain-containing adaptor protein) loci ablates all responses to LPS, indicating that only two signalling pathways emanate from the LPS receptor. However, a Trif-independent cell population is detectable when Trif(Lps2) mutant macrophages are stimulated with LPS. This reveals that an alternative MyD88-dependent 'adaptor X' pathway is present in some, but not all, macrophages, and implies afferent immune specialization.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antígenos de Diferenciação/fisiologia , Lipopolissacarídeos/farmacologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Antígenos de Diferenciação/genética , Escherichia coli/fisiologia , Homozigoto , Interferon Tipo I/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/virologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fator 88 de Diferenciação Mieloide , Fenótipo , Mapeamento Físico do Cromossomo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/genética , Análise de Sequência de DNA , Especificidade por Substrato , Receptor 3 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/metabolismo , Vaccinia virus/fisiologiaRESUMO
Macrophages are immune cells with high plasticity that perform many functions related to tissue injury and repair. They are generally categorized as 2 functional phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory and prohealing). To investigate the role of macrophages in human dental pulp, we examined the localization and distributional alterations of macrophages in healthy dental pulp as well as during the reparative process of pulp capping with mineral trioxide aggregate (MTA) and in cariously inflamed pulp of adult human teeth. We also quantified the populations of M1/M2 macrophages in healthy dental pulp by flow cytometric analysis. CD68+CD86+ cells (M1 phenotype) and CD68+CD163+ cells (M2 phenotype) were 2.11% ± 0.50% and 44.99% ± 2.22%, respectively, of 2.96% ± 0.41% CD68+ cells (pan-macrophages) in whole healthy dental pulp. Interestingly, M2 phenotype macrophages were associated with Schwann cells in healthy pulp, during mineralized bridge formation, and in pulp with carious infections in vivo. Furthermore, the M2 macrophages associated with Schwann cells expressed brain-derived neurotrophic factor (BDNF) under all in vivo conditions. Moreover, we found that plasma cells expressed BDNF. Coculture of Schwann cells isolated from human dental pulp and human monocytic cell line THP-1 showed that Schwann cells induced M2 phenotypic polarization of THP-1 cell-derived macrophages. The THP-1 macrophages that maintained contact with Schwann cells were stimulated, leading to elongation of their cell shape and expression of M2 phenotype marker CD163 in cocultures. In summary, we revealed the spatiotemporal localization of macrophages and potent induction of the M2 phenotype by Schwann cells in human dental pulp. M2 macrophages protect neural elements, whereas M1 cells promote neuronal destruction. Therefore, suppressing the neurodestructive M1 phenotype and maintaining the neuroprotective M2 phenotype of macrophages by Schwann cells may be critical for development of effective treatment strategies to maintain the viability of highly innervated dental pulp.
Assuntos
Polpa Dentária , Macrófagos , Células de Schwann , Capeamento da Polpa Dentária , Humanos , FenótipoRESUMO
It has been reported that there is a relationship between a single-nucleotide polymorphism (SNP) in the promoter region of the CD14 gene at position -159 (C-->T) and infectious diseases. The aim of the present study was to test the hypthesis that expression of this SNP correlates with periodontal disease in a Japanese population. The CD14 genotype was determined in 163 subjects with periodontitis and in 104 age- and gender-matched control subjects without periodontitis. The genotype distribution and allele frequency within the periodontitis patients were not significantly different from those of control subjects. There was, however, a significant difference in the genotype distribution between young patients (< 35 yrs) and older patients (>/==" BORDER="0"> 35 yrs). These findings suggest that CD14 -159C/T polymorphism is not related to the development of periodontitis in a Japanese population, but that, within the periodontitis subjects, expression of the SNP may be related to early disease activity.
Assuntos
Receptores de Lipopolissacarídeos/genética , Periodontite/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Adulto , Fatores Etários , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Citosina , Feminino , Frequência do Gene , Genótipo , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Periodontite/classificação , Porphyromonas gingivalis , Timina , Transcrição Gênica/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Early-onset periodontitis (EOP) is considered to have a genetic basis, which has not been clearly defined. The tumor necrosis factor-alpha (TNF-alpha) gene polymorphism as one of the genetic factors may influence the expression of several chronic inflammatory diseases. The aim of the present study was to evaluate whether the polymorphisms in the 5'-flanking region of the TNF-alpha gene are associated with Japanese EOP patients. METHODS: Forty-six Japanese, generalized EOP (G-EOP) patients and 104 Japanese healthy subjects were identified according to established clinical criteria. Twenty healthy subjects were analyzed by nucleotide sequence to screen polymorphisms of the 5'-flanking region of the TNF-alpha gene. Then, all subjects were analyzed by polymerase chain reaction and sequence-specific oligonucleotide probe (SSOP) methods. RESULTS: We determined 5 single nucleotide polymorphisms at positions -1031 (T/C), -863 (C/A), -857 (C/T), -308 (G/A), and -238 (G/A) in the 5'-flanking region of the TNF-alpha gene. There were no significant differences in the genotype and allele frequency when we compared G-EOP patients to healthy subjects. Because the frequency of polymorphic alleles at positions -308 and -238 was very low in this study population, we demonstrated the existence of 4 detected haplotypes and 6 detected genotypes concerning 3 single nucleotide polymorphisms (-1031, -863, and -857). The frequency of the H1/H3 (TCC/TCT)-detected genotype tended to decrease in G-EOP patients compared to healthy subjects, but was not statistically significant. CONCLUSION: These findings suggest there is no significant association between polymorphisms in the 5'-flanking region of the TNF-alpha gene and susceptibility to G-EOP in Japanese patients.
Assuntos
Região 5'-Flanqueadora/genética , Periodontite Agressiva/genética , Polimorfismo Genético/genética , Fator de Necrose Tumoral alfa/genética , Adulto , Alelos , Sequência de Bases/genética , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Feminino , Regulação da Expressão Gênica/genética , Frequência do Gene/genética , Predisposição Genética para Doença , Genótipo , Haplótipos/genética , Humanos , Japão , Masculino , Método de Monte Carlo , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Estatística como AssuntoRESUMO
Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca(2+) levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation.
Assuntos
Gengiva/citologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPV/fisiologia , Anilidas/farmacologia , Animais , Apoptose/fisiologia , Capsaicina/farmacologia , Linhagem Celular , Proliferação de Células , Células Cultivadas , Cinamatos/farmacologia , Células Epiteliais/fisiologia , Fatores de Crescimento de Fibroblastos/análise , Perfilação da Expressão Gênica , Gengiva/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/análise , Nociceptores/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Regulação para CimaRESUMO
INTRODUCTION: In addition to bacteria, viruses have been reportedly implicated in periodontitis. However, the available data are confined to Toll-like receptor 2 (TLR2) and TLR4, which recognize bacterial products in periodontitis. In the present study, we investigated the expression levels of TLR5, -7, and -9 messenger RNAs (mRNAs) in addition to those of TLR2 and -4, and compared gingivitis and periodontitis. Interferon-alpha1 (IFN-alpha1), which is important for the antiviral response, was also compared. METHODS: Gene expression was analyzed using quantitative real-time polymerase chain reaction for 59 periodontitis and 27 gingivitis tissue samples together with viral serology in some patients. The presence of plasmacytoid dendritic cells (pDCs), a robust producer of IFN-alpha, was immunohistochemically analyzed in an additional seven periodontitis and two gingivitis specimens. RESULTS: The expression levels of TLR2, -4, -7, and -9 were significantly higher in periodontitis lesions than gingivitis lesions. The expression level of TLR5 was comparable to levels of TLR2 and -4; however, no significant difference was found between gingivitis and periodontitis. Although the expression of IFN-alpha1 mRNA was higher in periodontitis lesions compared with gingivitis lesions, the level was quite low. Only a few pDCs were found in some periodontitis specimens. No difference was found for antibody-positivity between gingivitis and periodontitis. CONCLUSION: This is the first study to show that a variety of TLRs are up-regulated in periodontitis lesions compared with gingivitis lesions, suggesting that diverse microbial and possibly viral antigens are involved in the pathogenic mechanisms for periodontal diseases. However, the ligands recognized by the various TLRs in periodontal lesions remain to be determined.
Assuntos
Gengivite/imunologia , Interferon-alfa/análise , Periodontite/imunologia , RNA Mensageiro/análise , Receptores Toll-Like/análise , Adulto , Anticorpos Antivirais/sangue , Citomegalovirus/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Gengivite/patologia , Herpesvirus Humano 4/imunologia , Humanos , Imunoglobulina G/sangue , Interferon-alfa/genética , Lectinas Tipo C/análise , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Periodontite/patologia , Receptores Imunológicos/análise , Simplexvirus/imunologia , Receptor 2 Toll-Like/análise , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/análise , Receptor 4 Toll-Like/genética , Receptor 5 Toll-Like/análise , Receptor 5 Toll-Like/genética , Receptor 7 Toll-Like/análise , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/análise , Receptor Toll-Like 9/genética , Receptores Toll-Like/genéticaRESUMO
The identification of the TLRs as key sensors of microbial infection has presented a series of new targets for drug development. The TLRs are linked to the most powerful inflammatory pathways in mammals. The question arises from the start: do we wish to stimulate TLR signaling in order to eradicate specific infections and/or neoplastic diseases? Or do we wish to block TLR signaling to treat inflammatory diseases? If we accept that it would be useful to modulate TLR signaling, the next step is to identify the correct molecular target(s) for the task. Perhaps it might even be possible to exercise selectivity, modulating some aspects of TLR signaling and not others. Classical and reverse genetic analyses offer insight into the possibilities that exist, and point to specific checkpoints within signaling pathways at which modulation might normally be imposed.
Assuntos
Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/agonistas , Receptores Toll-Like/antagonistas & inibidores , Infecções Bacterianas/tratamento farmacológico , Receptores Toll-Like/imunologiaRESUMO
Recent evidence suggests that molecular mimicry between bacterial and human heat shock protein 60 (hsp60) is involved in various conditions of autoimmune and infectious diseases. Many periodontopathic bacteria have been reported to express GroEL-like protein that is homologous to human hsp60. In this study, the presence of antibodies to the hsp60 of Actinobacillus actinomycetemcomitans in the sera of periodontitis patients and periodontally healthy control subjects was evaluated by enzyme-linked immunosorbent assay using a recombinant A. actinomycetemcomitans GroEL as an antigen. Furthermore, their cross-reactivity with Escherichia coli GroEL and Mycobacterium bovis BCG hsp65 was examined. The mean values of antibody were 0.624 (range 0.088-1.113) and 0.728 (range 0.217-1.296) in control subjects and periodontitis patients, respectively. The antibody levels to A. actinomycetemcomitans after absorption with E. coli GroEL and M. bovis BCG clearly decreased in both control subjects and periodontitis patients. The remaining antibody levels to A. actinomycetemcomitans GroEL after absorption with M. bovis BCG hsp65 were higher than those with E. coli GroEL, indicating higher cross-reactivity with E. coli GroEL. These results suggest that not only periodontitis patients but also periodontally healthy subjects may be infected with A. actinomycetemcomitans but that the part of the antibody could be derived from the cross-reactivity with E. coli GroEL. Any relationship of the antibody to the disease, however, remains to be determined.
Assuntos
Aggregatibacter actinomycetemcomitans/química , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias , Chaperonina 60/imunologia , Periodontite/imunologia , Periodontite/microbiologia , Adulto , Estudos de Casos e Controles , Chaperoninas/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Escherichia coli/química , Feminino , Humanos , Masculino , Mycobacterium bovis/imunologia , Proteínas Recombinantes/imunologiaRESUMO
Heat shock protein 60 (hsp60) has been increasingly recognized as an important molecule in infectious and autoimmune diseases. We have demonstrated previously that serum antibodies to both human hsp60 and Porphyromonas gingivalis GroEL were elevated in periodontitis patients compared with healthy subjects. In order to clarify the relative importance of hsp60 in the inflammatory response in periodontal disease, the stimulatory effect of human and bacterial hsp60 on the production of tumour necrosis factor-alpha (TNF-alpha) was examined in phorbol myristate acetate (PMA)-stimulated THP-1 cells. As bacterial hsp60s, recombinant P. gingivalis and Actinobacillus actinomycetemcomitans GroEL was used. Human hsp60 but not P. gingivalis or A. actinomycetemcomitans GroEL demonstrated stimulatory activity similar to lipopolysaccharide (LPS) derived from the bacteria. The activity of hsp60 was inhibited by anti-CD14 and anti-Toll-like receptor 4 (TLR4) antibodies, suggesting that both CD14 and TLR4 mediate hsp60 signalling. Immunohistochemical analysis demonstrated that hsp60 is abundantly expressed in periodontitis lesions. Therefore, it is postulated that periodontopathic bacteria stimulate the cells in the periodontium to up-regulate the expression of hsp60, which in turn may stimulate macrophage and possibly other cells to produce proinflammatory cytokines. These mechanisms may be involved in the chronicity and tissue destruction of periodontal disease.
Assuntos
Chaperonina 60/metabolismo , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Carcinógenos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Chaperonina 60/imunologia , Chaperonina 60/farmacologia , Doença Crônica , Humanos , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Doenças Periodontais/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: IL-10 is an anti-inflammatory cytokine, which may modulate disease expression in chronic inflammatory periodontal disease. 3 dimorphic polymorphisms within the IL-10 gene promoter have recently been identified and appear to influence regulation of its expression. AIM: The aim of the present study was to investigate whether the promoter polymorphisms are associated with adult periodontitis (AP) and generalized early-onset periodontitis (G-EOP). METHODS: Genomic DNA was obtained from 34 AP patients, 18 G-EOP patients and 52 controls. The promoter region between -506 and -1140 was amplified by polymerase chain reaction, and polymorphisms were detected by nucleotide sequencing. RESULTS: The haplotype frequencies in Japanese were quite different from those of Caucasian and were even slightly different from those of southern Chinese with systemic lupus erythematosus. We found no significant difference in allele or haplotype frequencies between patients and controls. CONCLUSIONS: IL-10 production may be regulated within the complex cytokine network in chronic inflammatory periodontal disease, rather than the gene polymorphisms.
Assuntos
Haplótipos/genética , Interleucina-10/genética , Periodontite/genética , Polimorfismo Genético , Adulto , Alelos , Estudos de Casos e Controles , China/etnologia , Análise Mutacional de DNA , Feminino , Humanos , Japão/etnologia , Masculino , Regiões Promotoras Genéticas , População Branca/genéticaRESUMO
The presence of antibodies to the 60-kD human and Porphyromonas gingivalis GroEL hsp60 in the sera and inflamed gingival tissues of periodontitis patients was examined. In order to obtain the antigens, recombinant plasmids carrying human hsp60 and P. gingivalis GroEL genes were constructed and expressed as histidine-tagged recombinant proteins. Immunoreactivities of these proteins were confirmed by MoAbs specific to mammalian hsp60 and cross-reactive with both mammalian and bacterial hsp60. Western blot analysis clearly demonstrated that the number of periodontitis patients showing a positive response to P. gingivalis GroEL was higher than the number of periodontally healthy subjects. Furthermore, anti-P. gingivalis GroEL antibody was detected in all samples of gingival tissue extracts. For human hsp60, a higher frequency of seropositivity was found in the periodontitis patients than in the healthy subjects. In addition, the periodontitis patients demonstrated stronger reactivity compared with the healthy subjects. Quantitative analysis of serum antibodies by ELISA also demonstrated that the levels of antibodies in the sera of patients were significantly higher than those of control subjects. In the gingival tissue extracts, seven out of 10 patients demonstrated a positive response to human hsp60 and tso of these demonstrated strong positivity. Affinity-purified serum antibodies to human hsp60 and P. gingivalis GroEL from selected patients reacted with P. gingivalis GroEL and human hsp60, respectively, suggesting cross-reactivity of antibodies. These results suggest that molecular mimicry between GroEL of the periodontopathic bacterium P. gingivalis and autologous human hsp60 may play some role in immune mechanisms in periodontitis.
Assuntos
Chaperonina 60/imunologia , Periodontite/imunologia , Porphyromonas gingivalis/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Formação de Anticorpos/imunologia , Western Blotting , Chaperonina 60/biossíntese , Reações Cruzadas , Feminino , Gengiva/imunologia , Gengiva/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Periodontite/sangue , Periodontite/patologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Gingival fibroblasts produce proinflammatory cytokines in response to lipopolysaccharide (LPS) from periodontopathic bacteria. Recently it has become evident that the human homologue of Drosophila Toll can transduce intracellular signaling by LPS stimulation. Toll-like receptors (TLRs) have been identified in myeloid cells; however, their role in nonmyeloid cells such as gingival fibroblasts has not been fully elucidated. Here, we report that human gingival fibroblasts constitutively express TLR2 and TLR4 and that their levels of expression are increased by stimulation with LPS from Porphyromonas gingivalis. Upregulated expression of interleukin-6 gene and protein in fibroblasts stimulated with LPS is inhibited by anti-TLR4 antibody. These findings suggest that TLRs may confer responsiveness to LPS in gingival fibroblasts.
Assuntos
Proteínas de Drosophila , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/biossíntese , Porphyromonas gingivalis/patogenicidade , Receptores de Superfície Celular/biossíntese , Células Cultivadas , Fibroblastos/citologia , Gengiva/citologia , Humanos , Interleucina-6/biossíntese , Transdução de Sinais , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Regulação para CimaRESUMO
Chronic inflammatory periodontal diseases are characterized by a cellular infiltrate and are similar in many respects to other chronic inflammatory diseases. While periodontopathic bacteria have been recognized as the principal causative agent and the immune response to these bacteria is thought to be responsible for the tissue destruction, the full aetiological spectrum is still incompletely understood. In addition to many cell types such as polymorphonuclear leucocytes and macrophages, T cells have been implicated in pathogenesis and are considered to have regulatory roles in progression of the disease. Based on our recent studies demonstrating biased expression of several Vbeta families in periodontitis tissues, the aim of this study was to characterize further the T cells relevant to the disease process by reverse transcription-polymerase chain reaction-single-strand conformation polymorphism (RT-PCR-SSCP) and subsequent nucleotide sequence analysis of complementarity-determining region 3 (CDR3) of the TCR beta-chain. In spite of the likely involvement of numerous bacteria, the present study has clearly shown the oligoclonality of infiltrating T cells in periodontitis lesions in contrast to low clonality of peripheral blood T cells as evidenced by the appearance of distinct bands in gingival tissue samples and smear pattern of peripheral blood on SSCP gels. These were confirmed by the DNA sequencing of the CDR3 of Vbeta16 of selected samples. The analysis of deduced amino acid sequences demonstrated amino acid motifs in the CDR3 region of the periodontitis lesion-derived sequences from each patient. The results indicate that gingival tissue-infiltrating T cells recognizing a limited number of antigens or epitopes are involved in the disease process.